L E I B N I Z - I N S T I T U TF Ü R M O L E K U L A R E P H A R M A K O L O G I E

R E S E A R C H R E P O R T 2 011 / 2 012

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2

2 3

C O N T E N TS C I E N T I F I C A D V I S O R Y B O A R D

S C I E N T I F I CA D V I S O R Y B O A R D

C O N T E N T I N H A LT

Prof. Dr. Karl-Heinz AltmannInstitut für Pharmazeutische WissenschaftenETH Zürich (seit 01/2012)

Prof. Dr. Annette G. Beck-SickingerInstitut für Biochemie Universität Leipzig

Prof. Dr. Nils BroseMax-Planck-Institut für Experimentelle Medizin Göttingen(seit 01/2012)

Prof. Dr. Michael FreissmuthInstitut für PharmakologieUniversität Wien

Prof. Dr. Christian Griesinger (Vorsitzender)Max-Planck-Institut für Biophysikalische ChemieKarl-Friedrich-Bonhoeffer-InstitutGöttingen

Prof. Dr. Hans-Georg JoostDeutsches Institut für ErnährungsforschungPotsdam-Rehbrücke

Prof. Dr. Gerhard KlebeInstitut für Pharmazeutische ChemieUniversität Marburg

Prof. Dr. Frauke MelchiorZentrum für Molekulare Biologie Heidelberg (ZMBH)Ruprecht-Karls-Universität Heidelberg

Prof. Dr. Eckhard OttowBayer HealthCare PharmaceuticalsBerlin

Prof. Dr. Petra SchwilleMax-Planck-Institut für Biochemie Martinsried (seit 01/2012)

Prof. Dr. Herbert Waldmann Max-Planck-Institut für Molekulare PhysiologieDortmund(bis Ende 12/2011)

Stichtag 31.12.2012

Interview with Director Volker Haucke What’s New at the FMP?Was gibt es Neues am FMP? 4

Thomas J. JentschHow we Perceive SmellsWie wir Gerüche wahrnehmen 8

Andrew J. R. PlestedWhat Makes that Glutamate Receptor so Fast?Was macht den Glutamat-Rezeptor so schnell? 12

Leif SchröderDiagnosis with Xenon-AtomsDiagnose mit Xenon-Atomen 16

Volker HauckeBlockade in TransportBlockade im Transport 20

Leibniz Graduate School of Molecular Biophysics, Berlin 24

All Hands on Board: The Chemical Biology UnitMit vereinten Kräften: Die Chemical Biology Unit 25

NMR for the Whole of EuropeNMR für ganz Europa 28

Structural Biology SectionBereich Strukturbiologie 31

NMR-supported Structural Biology Hartmut Oschkinat 34Protein Engineering Christian Freund 38Structural Bioinformatics and Protein Design Gerd Krause 42Computational Chemistry / Drug Design Ronald Kühne 46Solution NMR Peter Schmieder 50Molecular Imaging Leif Schröder 54In-Cell NMR Philipp Selenko 58

Molecular Physiology and Cell Biology SectionBereich Molekulare Physiologie und Zellbiologie 63

Physiology and Pathology of Ion Transport Thomas J. Jentsch 66Molecular Cell Physiology Ingolf E. Blasig 70Molecular Pharmacology and Cell Biology Volker Haucke 74Behavioural Neurodynamics Tatiana Korotkova /Alexey Ponomarenko 78Molecular Neuroscience and Biophysics Andrew J. R. Plested 82Protein Trafficking Ralf Schülein 86Cellular Imaging / Electron Microscopy Burkhard Wiesner 90

Chemical Biology SectionBereich Chemische Biologie 95

Chemical Biology II Christian P. R. Hackenberger 98Peptide Chemistry Michael Beyermann 102Peptide-Lipid Interaction / Peptide Transport Margitta Dathe 106Chemical Systems Biology Ronald Frank 110Mass Spectrometry Eberhard Krause 114Screening Unit Jens Peter von Kries 118Protein Chemistry Dirk Schwarzer 122

Administrative and Technical Services 126 Index 127 Map Campus Berlin Buch 130Imprint 132

P R E FA C EV O R W O R T

R E S E A R C H H I G H L I G H T SA K T U E L L E S A U S D E R F O R S C H U N G

R E S E A R C H G R O U P SF O R S C H U N G S G R U P P E N

4 5

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2W H AT ‘ S N E W AT T H E F M P ? W A S G I B T E S N E U E S A M F M P ?

Together with your very successful research group, you moved from the Freie Universität Berlin to the FMP. This means a considerable upheaval for all concerned, and you yourself have to put up with a long journey to work every day. What is special about the FMP, what appeals to you about the new environment?The FMP offers very special opportunities for interdisciplinary co-operation between biologists, chemists and physicists: Here, we can explore physiological processes and the effect of genetic constellations all the way down to the structure of individual molecules, proteins and their ligands. At the same time, the FMP has always been a motor for technological developments, which in turn lead to advancements in biological research. Together with my research group, I would like to make a contribution here, too, e.g. by further developing the technique of high-resolution fluorescence microscopy. This technique can be used to visualise processes in cells and tissue down to molecular detail, and the conditions available at a non-university institute like the FMP are particularly good for further developing this kind of advanced technology.

The research at FMP is orientated towards discovering new active substances that might be used as a basis for medicinal products in the future. What does the Institute have to offer that is not already provided by university and commercial pharmacological research?Traditional pharmacological research often investigates already known substances or their derivatives and their effect in a physi-ological context. A radical approach like the one taken here at the FMP of developing novel active substances on the basis of biological structures of proteins or protein complexes and their ligands, would be almost impossible to implement at universi-ties. And industrial concerns are mainly interested in active sub-stances that are orientated towards established structures that are known to be related to the major diseases of our times. Our approach is generally the other way around: We are investigating

the fundamental molecular mechanisms of cells and organisms, in the hope of discovering target structures that have not been detected to date, for which novel active substances then can be developed. The outcome is naturally far more uncertain in such an approach, but there is a potential to make groundbreaking discoveries.

What were the greatest successes achieved by the FMP over the past two years?I can only mention a few of the numerous successful publica-tions here. For example, Thomas Jentsch’s group identified the first human gene that has an effect on the still little understood sense of touch. It codes for an ion channel in nerve cells that is responsible for the fine-tuning of touch. One fascinating aspect is that this channel also plays a role in hearing, and mutations here lead to deafness.

The group led by Barth van Rossum has succeeded in visualis-ing the surface molecule of a bacterial pathogen that causes diarrhoea and inflammatory diseases. In co-operation with the Max Planck Institute for Developmental Biology in Tübingen, the group used solid-state NMR to determine the structure of an important part of this molecule, which plays an important role in the process of infection.

Phil Selenko and his co-workers used high-resolution NMR spec-troscopy to observe how chemical tags are attached to a histone molecule, and thus managed to decipher some of the code ex-pressed by these tags. Histones are in effect the spools around which our DNA is wound, and the tags decide which genes are read at all. Defects in “writing” or “reading” these tags can lead to serious diseases such as cancer.

Together with his colleagues, Andrew Plested succeeded in im-proving our understanding of the molecular machinery of the glutamate receptor. This receptor, which is immensely important for our nervous system, has to be fully activated within a thou-sandth of a second in order to be able to act reliably. Andrew effectively took the receptor apart and then put the modules of different types back together again, thus revealing which module is responsible for the unbelievably rapid reaction.

And my own group, together with the FMP’s Screening Unit, has developed small molecules that are capable of blocking a central transport process through which cells take up signal molecules or also pathogens. These so-called “pitstops” bind to certain pro-

teins, through which the cells form invaginations, a structure that had been inaccessible to active substances to date. The transport process is of fundamental importance and, building on our find-ings, we hope one day to find new concepts for treating diseases such as viral infections that have been difficult to treat to date.

Beside this and other work, we have initiated three large projects that are designed to coordinate our research more closely with other European institutes. The fundamental idea behind this is to combine complicated technology and expertise that is too exten-sive for an individual institute to shoulder on its own. Through “In-struct”, the FMP is now integrated in a European network in which the different technologies are applied concertedly for the elucida-tion of biological structures. We are additionally involved in the EU project “Bio-NMR”, through which other European groups have access to our high-performance NMR devices. And the FMP is in charge of developing the project “EU-OPENSCREEN”, which has just been given a positive appraisal. This is aimed at coordinating the search for active substances throughout Europe.

What has changed under your leadership and what further changes are to be expected?We have a new postgraduate programme, which is designed to improve scientific exchange and contact between the groups. And we have pulled off a terrific appointment: Prof. Christian Hackenberger has taken on the Leibniz-Humboldt chair for Chemical Biology – he is engaged in research into the synthesis and modification of proteins and peptides. In addition, we are looking forward to new junior research groups, especially since they are going to be led by women: Janine Kirstein-Miles, who will be leading an independent junior research group from the summer of 2013 onwards, uses nematodes to understand mecha-nisms with which protein aggregates can be dissolved. Such aggregates occur e.g. in neurodegenerative disorders such as Alzheimer’s disease and are also of great importance in gerontol-ogy. Tanja Maritzen will also be leading a group of her own within my department and will be receiving special funding, which she was awarded as part of a competition within the Leibniz institutes. And finally, we are looking forward to welcoming the outstanding medicinal chemist Marc Nazaré into our ranks at the FMP, whose role will be to synthesise customised active substances for us. He belongs to the overall concept of the “Chemical Biology Unit”, with which the development of new active substances is to be given extra impetus. As a whole, the FMP now has so many out-standing scientists at its disposal that I am looking forward to a very exciting future.

The FMP has a new Director: Since the beginning of 2012, the pharmacologist and bio-chemist Prof. Dr. Volker Haucke has been in charge of the Institute. In an interview, he explains what he considers to be special about the Institute, what outstanding achieve-ments there have been over the past few years, and what changes have been made and are still to be expected under his leadership.

Sie sind mit Ihrer sehr erfolgreichen Arbeitsgruppe von der Freien Universität Berlin ans FMP gewechselt. Das bedeutet für alle eine große Umstellung, Sie selbst nehmen nun täglich eine lange Anfahrt in Kauf. Was ist das Besondere am FMP, was hat Sie an dem neuen Umfeld gereizt?Das FMP bietet ganz besondere Möglichkeiten der interdiszipli-nären Zusammenarbeit von Biologen, Chemikern und Physikern: Wir können hier physiologische Vorgänge und die Auswirkung genetischer Konstellationen bis hin zur Struktur einzelner Molekü-le, der Proteine und ihrer Liganden ergründen. Zugleich war das FMP schon in der Vergangenheit ein Motor für technologische Entwicklungen, die wiederum biologische Forschung voranbrin-gen. Mit meiner Arbeitsgruppe möchte auch ich hier einen Bei-trag leisten, z.B. indem wir die Technik der hochauflösenden Fluo-reszenzmikroskopie weiterentwickeln. Mit ihrer Hilfe kann man die Vorgänge in Zellen und Geweben bis ins molekulare Detail sicht-bar machen, und in einem außeruniversitären Institut wie dem FMP hat man besonders gute Bedingungen, eine solche Hoch-technologie voranzubringen.

Die Forschung am FMP ist darauf ausgerichtet, neue Wirkstoffe zu erforschen, aus denen zukünftige Medikamente hervorgehen können. Was kann das Institut leisten, was nicht schon univer-sitäre und kommerzielle pharmakologische Forschung bietet?Die traditionelle pharmakologische Forschung untersucht oftmals schon bekannte Substanzen oder deren Derivate und ihre Wir-kung im physiologischen Zusammenhang. Ein radikales Vorgehen wie hier am FMP, auf der Basis biologischer Strukturen von Prote-inen oder Proteinkomplexen und ihren Liganden ganz neue Wirk-stoffe zu entwickeln, ist an Universitäten nur schwer möglich. Und

Prof. Dr. Volker Haucke

Director of Leibniz-Institut für

Molekulare Pharmakologie

WHAT‘S NEW AT THE

FMP?

Das FMP hat einen neuen Direktor: Seit Anfang 2012 leitet der Pharmakologe und Biochemi-ker Prof. Dr. Volker Haucke das Institut. Im Interview er-zählt er, was für ihn das Besondere des Instituts ausmacht, welche herausragenden Leistungen es in den letzten Jah-ren gegeben hat, und welche Veränderungen es unter seiner Leitung gab und noch geben wird.

W A S G I B T E S N E U E S

A M F M P ?

6 7

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2 W I E W I R G E R Ü C H E W A H R N E H M E NW H AT ‘ S N E W AT T H E F M P ?

die Industrie sucht vor allem nach Wirkstoffen, die auf bekannte Strukturen abzielen, von denen man weiß, dass sie mit den gro-ßen Volkskrankheiten in Zusammenhang stehen. Unsere Vorge-hensweise ist in der Regel umgekehrt: Wir erforschen die grund-legenden molekularen Mechanismen von Zellen und Organismen, in der Hoffnung, bisher unbekannte Zielstrukturen aufzuspüren, für die dann neuartige Wirkstoffe entwickelt werden können. Bei einem solchen Vorgehen ist der Ausgang natürlich viel ungewis-ser, dafür kann so bahnbrechend Neues entstehen.

Über welche Erfolge konnte sich das FMP in den letzten bei-den Jahren besonders freuen?Ich kann hier nur einige der zahlreichen erfreulichen Publikationen erwähnen. Zum Beispiel hat die Gruppe von Thomas Jentsch das erste menschliche Gen identifiziert, das sich auf den noch wenig verstandenen Tastsinn auswirkt. Es kodiert für einen Ionenkanal in Nervenzellen, der für das „Feintuning“ des Tastens verantwort-lich ist. Faszinierenderweise spielt dieser Kanal auch beim Hören eine Rolle, und Mutationen führen hier zur Taubheit.

Der Gruppe von Barth van Rossum ist es gelungen, das Ober-flächenmolekül eines bakteriellen Krankheitserregers darzustel-len, der Durchfälle und entzündliche Erkrankungen verursacht. In Zusammenarbeit mit dem Max-Planck-Institut für Entwicklungs-biologie in Tübingen hat die Gruppe mittels Festkörper-NMR die Struktur eines wichtigen Teils dieses Moleküls aufgeklärt, das bei der Infektion eine entscheidende Rolle spielt.

Phil Selenko und seine Mitarbeiter haben mit hochauflösender NMR-Spektroskopie beobachten können, wie die chemischen Markierungen an einem Histon-Molekül angebracht werden, und so den zugrundeliegenden Code für ihre Bedeutung teilweise entschlüsselt. Histone sind quasi die Spulen, um die unsere DNA gewickelt wird, und die Markierungen entscheiden darüber, wel-che Gene überhaupt abgelesen werden. Defekte beim „Schrei-ben“ oder „Ablesen“ der Markierungen können zu schweren Krankheiten wie zum Beispiel Krebs führen.

Andrew Plested ist es mit seinen Kollegen gelungen, die moleku-lare Maschinerie des Glutamatrezeptors ein Stück besser zu ver-stehen. Dieser für unser Nervensystem immens wichtige Rezep-tor  muss, um verlässlich agieren zu können, innerhalb einer tausendstel Sekunde vollständig aktiviert sein. Andrew hat den Rezeptor quasi auseinandergenommen und die Module unter-schiedlicher Typen neu zusammengesetzt, und konnte so zeigen, welches Modul für die unglaubliche Schnelligkeit verantwortlich ist.

Und meine eigene Gruppe hat zusammen mit der Screening Unit des FMP kleine Moleküle entwickelt, die einen zentralen Trans-portvorgang blockieren können, durch den Zellen Signalmolekü-le oder auch Krankheitserreger aufnehmen. Diese sogenannten

„Pitstops“ binden an bestimmte Proteine, durch die Zellen Einstül-pungen formen, eine bislang für Wirkstoffe unzugängliche Struk-tur. Der Transportvorgang ist von fundamentaler Bedeutung, und vielleicht werden wir darauf aufbauend einmal neue Konzepte zur Behandlung bislang schwer therapierbarer Krankheiten wie z.B. viraler Infektionen finden.

Neben diesen und anderen Arbeiten haben wir drei große Pro-jekte auf den Weg gebracht, um unsere Forschung enger mit

der anderer europäischer Institute zu verzahnen. Der Grundge-danke ist dabei, aufwändige Technik und Expertise zu kombinie-ren, über die kein Institut allein verfügen kann. Durch „Instruct“ ist das FMP nun in ein europäisches Netzwerk eingebunden, in dem die unterschiedlichsten Technologien konzertiert zur Aufklä-rung biologischer Strukturen eingesetzt werden. Wir sind außer-dem an dem EU-Projekt „Bio-NMR“ beteiligt, durch das andere europäische Gruppen Zugang zu unseren sehr leistungsstarken NMR-Geräten erhalten. Und das FMP entwickelt federführend das Projekt „EU-OPENSCREEN“, das gerade erfolgreich begutach-tet wurde. Darin soll die Suche nach neuen Wirkstoffen europa-weit koordiniert werden.

Was hat sich unter Ihrer Leitung bereits getan und welche Ver-änderungen wird es noch geben?Wir haben nun ein neues Doktorandenprogramm, durch das der wissenschaftliche Austausch und der Kontakt zwischen den Grup-pen verbessert wird. Und uns ist eine grandiose Berufung gelun-gen: Prof. Christian Hackenberger hat die Leibniz-Humboldt-Pro-fessur für Chemische Biologie übernommen. Er beschäftigt sich mit der Synthese und Modifikation von Proteinen und Peptiden. Außerdem freuen wir uns auf neue Nachwuchsgruppen, ganz besonders, weil sie von Frauen geleitet werden: Janine Kirstein-Miles, die ab dem Sommer 2013 eine unabhängige Nachwuchs-gruppe leiten wird, benutzt Fadenwürmer, um Mechanismen zu verstehen, mit denen Proteinaggregate aufgelöst werden können. Diese kommen z.B. in neurodegenerativen Krankheiten wie Mor-bus Alzheimer vor und sind auch in der Altersforschung von gro-ßer Bedeutung. Tanja Maritzen wird innerhalb meiner Abteilung ebenfalls ihre eigene Gruppe leiten und erhält dafür besondere Fördermittel, die sie in einem Wettbewerb innerhalb der Leibniz-Institute gewinnen konnte. Und schließlich freuen wir uns, dass bald auch der hervorragende Medizinalchemiker Marc Nazaré zum FMP gehören wird, der für uns maßgeschneiderte Wirkstof-fe synthetisieren wird. Er gehört zum Gesamtkonzept der „Che-mical Biology Unit“, mit der die Entwicklung neuer Wirkstoffe vor-angetrieben wird. Insgesamt ist das FMP nun mit hervorragenden Wissenschaftlern so gut aufgestellt, dass ich mich auf eine sehr spannende Zukunft freue.

RESEARCHHIGHLIGHTS

8 9

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2H O W W E P E R C E I V E S M E L L S W I E W I R G E R Ü C H E W A H R N E H M E N

To have a closer look on how the lack of signal amplification by chloride channels in these mice affects odor signaling, Balázs Pál measured electroolfactograms in which he determined the electrical response of many olfactory neurons to an odorant. For electroolfactogram recordings the olfactory mucosa is dis-sected from mice and placed in a small chamber with nutrient solution. While the tissue is continuously superfused with buffer, the electrical response to different odorants can be measured by placing a fine glass electrode on the surface of the epithelium. Comparing wild-type and Ano2 knock-out mice the researchers made another surprising observation: the olfactory answer of the knock-out variant was barely reduced. Regardless of the type of fragrance, whether flowery odour mixtures or fear-provoking coyote urine, the olfactory epithelium produced electrical signals that even without Ano2 still achieved at least 60 per cent of the normal values.

But how would loss of Ano2 influence the sense of smell in the living animal? Gwendolyn Billig went on to study her mice in be-

havioural experiments. For these olfactometric tests, she trained knock-out and wild-type animals to receive their drinking water through a special device from which water was only available after presentation of an odour. She took care to use mouse pairs from the same litter thereby minimizing genetic variance and exclud-ing differences in behavioural experience. In the actual smell test, two different odours are presented to the mice in random order, but only one of the odours is associated with a water reward. If mice are able to smell and thus can discriminate between the two odours they will soon stop licking in response to the non-re-warding odour. This behavioural change can be directly recorded and analysed as the water delivery device is coupled to a light barrier and an electrical contact. There are 10 million olfactory neurons in the mouse nose and to challenge them, Gwendolyn Billig devised tasks of increasing difficulty. First, the animals had to learn to discriminate between the fruity octanal and the chemi-cally similar hexanal which smells of freshly mown grass. In more complex tasks she trained the animals to differentiate between mixtures of both odours in differing fractions. She also deter-mined the detection limit for geraniol, the primary component of rose oil, to see if odour sensitivity was affected. Yet, there was no difference. Even in the most difficult tasks the Ano2 knock-out mice performed as well as their control littermates. Their sense of smell seemed totally normal – even without Ano2.

Apparently, the prominent paradigm of olfactory signal transduc-tion is wrong in one point. The chloride channel exists but its role

Even in the most difficult tasks the Ano2 knock-out mice performed as well as their control littermates. Their sense

of smell seemed totally normal – even without Ano2.

In a section of a mouse nose the

calcium-activated chloride channel

Ano2 has been made visible by

labeling with an antibody (green).

The Ano2 protein is found in the

thin layer of sensory cilia that cover

the surface of the main olfactory

epithelium (top) and the pheromone-

sensing vomeronasal organ (bottom).

Blue structures represent cell nuclei.

The group of Thomas Jentsch has succeeded in identifying the long sought-after chloride channel of olfaction and found a surprise: olfactory perceptions do not evolve in the way described in the textbooks.

It was a tight international competition in which Thomas Jentsch’s lab was hunting an unknown: a chloride channel in the olfactory mucosa thought to be vital for the sense of smell. Even though the channel’s activity was first described over 20 years ago the underlying gene had never been found. Only in 2008, with the identification of a novel family of ion channels, a promising fresh trail emerged. Following this track the researchers in Thomas Jentsch’s group were the first to identify Ano2 as the long sought-after chloride channel of sensory neurons of the nose. Yet, their hunt ended with a startling outcome: contrary to what one can read in many physiology textbooks, the channel is not necessary for normal olfaction.

As for all senses, the nose needs to convert an environmental cue into an electrical nerve signal. When we smell, with every breath odour molecules enter our nose and bind to receptors in the olfactory mucosa. These receptors are sitting on extremely fine processes of the olfactory neurons, the so-called sensory cilia that reach out from the neuron’s head into the nasal mucus. Out of the hundreds of possible receptors each neuron exposes on its cilia only one type of receptor that is specific for a certain odorant. Its activation induces an electrical signal in the nerve which travels to the olfactory brain where it is further processed and relayed to higher brain centres. Depending on the combination of activated nerve cells we will perceive such different odours as the smell of roses, of fried chicken or of stale sweat.

In the olfactory cilia, binding of an odour molecule to an olfactory receptor triggers a signalling cascade inside the sensory cell: the level of the second messenger cAMP rises, thereby opening so-called CNG (“cyclic nucleotide-gated”) channels. This allows posi-tively charged sodium and calcium ions to enter the cell, resulting

in depolarization of the membrane. According to the classical olfactory model, the inflowing calcium then activates a second channel through which negatively charged chloride ions exit the cell. This chloride current is believed to greatly amplify the initial depolarization – and has up to now been considered essential for normal olfactory function.

“We had suspected for some time that the membrane protein Ano2 might be the decisive chloride channel in the nerve cells of the olfactory mucosa,” says Thomas Jentsch. “Ano” is the abbre-viation for the gene family name “anoctamin”, a merge from the words “anions” for the anion selectivity of the channels and “octa” for the eight sections with which the protein spans the membrane. However, the definite proof was only made possible when Gwen-dolyn Billig, at that time a doctoral student in the group, gener-ated genetically modified knock-out mice in which the Ano2 gene is destroyed. Using specific antibodies she first confirmed that the Ano2 protein occurs indeed at very high levels in the olfactory mu-cosa. Besides the nose, she found Ano2 only in the eyes, the olfac-tory bulb and at very low levels in a few other parts of the brain. As expected for the olfactory chloride channel, fluorescent labelling of Ano2 showed that in normal mice it is located on the cilia of sensory neurons, while in the knock-out mice Ano2 was missing.

Balázs Pál, a postdoctoral scientist in the Jentsch group, con-ducted the critical key experiment. He investigated the chloride channel in single olfactory neurons by increasing the calcium con-centration inside the cell and measuring the chloride current over the cell membrane using the patch-clamp method. As expected, he found that in wild-type mice, upon activation with calcium, chloride exits olfactory neurons. However, no such chloride cur-rent could be detected when the Ano2 gene was deleted. Thus, his measurements gave the final proof: Ano2 is indeed the long sought-after chloride channel.

So what happens to the sense of smell of mice when Ano2 is miss-ing? Is the signal amplification by chloride channels as crucial to olfaction as expected? With the Ano2 knock-out mice in hand the scientists finally had a way to investigate the importance of calcium-activated chloride channels for olfactory processing. The first surprise when analysing the Ano2 knock-out mice came from simply looking at them. Mouse pups depend on olfactory cues to find their mother’s teats and thus need a functional sense of smell to feed and to survive. However, these animals thrived normally and were indistinguishable from their wild-type littermates casting initial doubts on Ano2’s importance for olfaction.

H O W W E P E R C E I V E S M E L L S

W I E W I R G E R Ü C H E W A H R N E H M E N

T H O M A S J . J E N T S C H

“We had suspected for some time that the membrane protein Ano2 might be the decisive chloride

channel in the nerve cells of the olfactory mucosa,” says Thomas Jentsch.

10 11

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2H O W W E P E R C E I V E S M E L L S W I E W I R G E R Ü C H E W A H R N E H M E N

der Präparate gab es wieder eine Überraschung: Die Reaktion der Knock-out-Variante war im Vergleich zu den normalen Mäu-sen kaum vermindert.

Egal, ob es sich um blumige Düfte oder um den für Mäuse furcht-erregenden Geruch von Kojoten-Urin handelte, das Riechepithel produzierte elektrische Signale, die auch ohne Ano2 immerhin noch 60 Prozent der normalen Werte erreichten. Und als die Duft-stoffe nicht in Lösung, sondern gasförmig – wie es physiologisch realistischer ist – verabreicht wurden, war der Unterschied zwi-schen normalen Mäusen und Mutanten sogar ganz verschwunden.

Um noch besser einschätzen zu können, wie gut Mäuse ohne Chloridkanal in den Sinneszellen riechen können, trainierte Gwen-dolyn Billig schließlich Mäuse darauf, Gerüche zu unterscheiden. Sie testete dabei Knock-out-Mäuse, die zusammen mit den Ver-gleichstieren von der gleichen Mutter aufgezogen worden waren, um unterschiedliche Erfahrungen und genetische Faktoren weit-gehend auszuschließen. Die Mäuse erhielten ihr Trinkwasser zeit-weise durch eine kleine Vorrichtung, in der sie mit der Schnauze eine Lichtschranke durchbrechen und einen Wassertropfen auf-lecken können. Durch einen elektrischen Kontakt kann man mes-sen, ob eine Maus leckt oder nicht. Die Mäuse mussten nun ler-nen, dass es Wasser nur bei einem bestimmten Duft gab, während das Lecken bei einem anderen Geruch sinnlos war. Zehn Millio-nen Riechzellen besitzen Mäuse in ihrem Riechepithel, und um die Nager so richtig zu fordern, erdachte Billig verschiedene Auf-gabenstellungen. Ganz schnell lernen Mäuse zum Beispiel, fruch-tig riechendes Ocatanal von Hexanal zu unterscheiden, dessen Geruch eher an frisch gemähtes Gras erinnert. Schwieriger kann man es dagegen für die Tiere machen, indem man Mischungen aus beiden Duftstoffen präsentiert, die sich lediglich in ihren Anteilen unterscheiden, oder wenn man generelle Duftstoffe immer weiter verdünnt. Doch auch bei den schwierigsten Aufga-ben, für die auch normale Mäuse viele Lerneinheiten benötigen, zeigte sich: Die Knock-out-Mäuse meisterten die Aufgaben auch

ohne Chlorid-Ausstrom aus den Riechzellen genauso gut wie ihre Verwandten mit funktionierendem Ano2.

Offensichtlich ist also das bisherige Modell in einem Punkt falsch. Der Chlorid-Ausstrom, ein oft postuliertes Musterbeispiel für eine Signalverstärkung in Nervenzellen, existiert zwar, doch auch ohne ihn können Mäuse ausgezeichnet riechen und entwickeln sich nor-mal. Doch wofür ist Ano2 dann überhaupt gut, und wie war man zu der falschen Vorstellung gekommen? „Die ersten Untersu-chungen wurden an Fröschen gemacht“, erklärt Thomas Jentsch. Amphibien und allgemein Süßwassertiere brauchen im Gegen-satz zu Säugetieren auf jeden Fall den Chlorid-Ausstrom aus den Riechzellen, denn das elektrisches Signal kann nicht durch ein-strömendes Natrium entstehen – dafür ist im Süßwasser schlicht zu wenig Natrium vorhanden. Spätere Untersuchungen an Säuge-tieren wurden vor allem an isolierten Nervenzellen gemacht. Hier könnte die empfindliche Struktur der Nervenzellen mit ihren lan-gen dünnen Zilien zu falschen Ergebnisse geführt haben.

Wäre es möglich, dass Ano2 gar keine biologische Funktion hat, sondern nur ein Überbleibsel aus der Amphibienzeit in unseren Genen ist? „Das ist unwahrscheinlich, ich glaube es eigentlich nicht“, meint Thomas Jentsch. „Schließlich genügt schon eine Punktmutation, damit der Chloridkanal nicht mehr funktioniert – so lange wäre er wohl also kaum konserviert geblieben.“

Die Rolle von Ano2 bleibt vorerst ein Rätsel. „Vielleicht ist er für die sehr feine Einstellung der Geruchswahrnehmung wichtig, etwa bei der Adaption an bestimmte Gerüche oder für sehr schnelle Reak-tionen – Eigenschaften, die nur unter ganz bestimmten Bedingun-gen wichtig sind“, spekuliert Gwendolyn Billig. Denkbar ist auch, dass er in Kombination mit dem im Körper viel häufiger vorhan-denen Chloridkanal Ano1 ein Rolle spielt. Beide Kanäle werden in Säugetieren gemeinsam in einem speziellen Bereich im Nasen-raum, dem Vomeronasalen Organ exprimiert, über das zum Bei-spiel Pheromone, also Signale von Artgenossen, wahrgenommen werden, oder auch Geruchssignale anderer Arten wie Fressfeinden. Thomas Jentsch plant derzeit Versuche mit Mäusen, in denen Ano1 und Ano2 zugleich ausgeschaltet sind. Die Geschichte des Chlo-ridkanals im Riechorgan bleibt also weiter spannend.

Die Knock-out-Mäuse meisterten die Aufgaben auch ohne Chlorid-Ausstrom aus den Riechzellen genauso gut

wie ihre Verwandten mit funktionierendem Ano2.

Billig GM, Pál B, Fidzinski P, Jentsch TJ (2011). Ca2+-activated Cl- channels are dispensable for olfaction. Nat Neurosci 14: 763-749.

has been largely overestimated in the past: olfactory detection works just fine without Ano2. But what is the benefit of having Ano2, and why had it been assigned such a prominent role in olfac-tion? “The first investigations were conducted on frogs,” explains Thomas Jentsch. In contrast to mammals, amphibians absolutely depend on a chloride efflux from the olfactory neurons for olfac-tory signal generation, since in fresh water there is too little sodium to support generation of the electrical signal solely by inflowing sodium. Another point might be that most of the investigations on mammals were conducted on isolated olfactory neurons. Their extremely sensitive structure could become damaged during the isolation process thus changing major physiological properties and possibly leading to false results.

Could it be that Ano2 has no biological function at all and is only a genetic vestige from amphibian times? “That is improbable, and I don’t think that’s the case,” says Thomas Jentsch. “After all, just one point mutation is enough to destroy a channel’s function – why should it then have been conserved for so long?”

The role of Ano2 remains a mystery for the time being. “Perhaps it is important for the fine adjustment of olfactory perception, such as adaptation to certain odours or for very rapid reactions – characteristics that are only important under very specific con-ditions that we did not test,” speculates Gwendolyn Billig. It is also conceivable that it plays a role in the vomeronasal organ where the Ano2 channel operates together with its close rela-tive Ano1. This specialized organ in the mouse olfactory system is important for sensing signals secreted by other animals, such as sex pheromones or predator odours. Thomas Jentsch is cur-rently planning experiments with mice in which Ano1 and Ano2 are inactivated at the same time. Thus, the story of the chloride channel in olfaction will keep us in suspense for a while longer.

Es war ein Unbekannter, auf den weltweit gleich mehrere Arbeits-gruppen Jagd machten: Ein Chloridkanal in der Riechschleimhaut, so nahm man an, müsste bei allen Säugetieren für den Geruchs-sinn unentbehrlich sein. Niemand jedoch wusste etwas Genaue-res über das vermutete Membranprotein. Erst als 2008 eine neue Gen-Familie von Ionenkanälen identifiziert und kloniert wurde, gab es eine konkrete Fährte. Die Jagd endete allerdings mit einem verblüffenden Ergebnis. Denn der Ionenkanal „Ano2“ sorgt zwar tatsächlich dafür, dass in der Nase bei einer Aktivierung der Sin-neszellen Chlorid ausströmt – doch notwendig für die Geruchs-wahrnehmung ist dies entgegen bisheriger Lehrmeinung nicht.

Nach dieser Entdeckung lässt sich nun ein präziseres Bild zeich-nen, wie Gerüche unser Gehirn erreichen. Wie bei allen Sinnen müssen auch beim Riechen Informationen über die Umwelt in elektrische Nervensignale umgewandelt werden. Herangewirbelt mit jedem Atemzug, binden einzelne Moleküle an Rezeptoren in der Riechschleimhaut. Hier enden die Riechzellen und ragen mit ihren hauchdünnen Fortsätzen, den Zilien, in die Schleimschicht

„Wir ahnten schon länger, dass das Membran- protein Ano2 der entscheidende Chloridkanal in den

Nervenzellen der Riechschleimhaut sein könnte“

hinein. Jede Zelle bildet auf ihren Zilien nur einen von hunderten möglichen Rezeptoren aus, der spezifisch ein bestimmtes Duft-molekül binden kann. Die dadurch erzeugten elektrischen Signa-le wandern die Nervenfasern entlang ins nahegelegene Riechhirn. Je nach Kombination von aktivierten Nervenzellen erleben wir so unterschiedliche Wahrnehmungen wie den Geruch von Rosen, von Brathähnchen oder altem Schweiß.

Bindet ein Molekül an einen Geruchsrezeptor, wird in der Sin-neszelle eine Signalkaskade eingeleitet: Der Botenstoff cAMP öffnet die sogenannten CNG („cyclic nucleotide-gated“)-Kanä-le, dadurch können Natrium und Calcium in die Zelle eindrin-gen, ihr Ruhepotential wird depolarisiert. Das einströmende Calcium aktiviert wiederum einen weiteren Kanal, durch den in den Zellen angereichertes Chlorid ausströmen kann. Nur durch diese Verstärkung, so glaubte man bislang, kommt es zu dem Nervensignal.

„Wir ahnten schon länger, dass das Membranprotein Ano2 der entscheidende Chloridkanal in den Nervenzellen der Riech-schleimhaut sein könnte“, erinnert sich Thomas Jentsch an die spannende Zeit, als die Gruppe sich daran machte, den Beweis zu erbringen. Der Name „Ano“ ist die Abkürzung für den Gen-Familiennamen „Anoctamine“, das Wort setzt sich zusammen aus

„Anionen“ und „octa“ für die meist acht Abschnitte, mit denen die Proteine die Membran durchspannen. Gwendolyn Billig, zu der Zeit Doktorandin in der Gruppe, gelang es schließlich als Erste, genetisch veränderte Knock-out-Mäuse ohne funktionie-rendes Ano2 zu erzeugen.

Von Anfang an fiel auf, dass diese Mäuse überraschend normal und gesund wirkten. Bei Mäusen ist der Geruchssinn absolut lebenswichtig; ohne ihn finden schon die Jungen nicht die Milch-zitzen ihrer Mutter und verhungern. Durch Anfärbungen mit spe-zifischen Antikörpern konnte Gwendolyn Billig aber bestätigen: Ano2 kommt fast ausschließlich in der Riechschleimhaut und im Auge vor, kaum dagegen im Gehirn, und hier vor allem im Riech-kolben. Fluoreszenzaufnahmen zeigten den Rezeptor bei norma-len Mäusen auf der Oberfläche von Riechzellen, bei den Knock-out-Mäusen war er wie erwartet verschwunden.

Als nächstes untersuchte Balázs Pál, ein Wissenschaftler aus der Jentsch-Gruppe, wie sich Ano2 in isolierten Neuronen verhält. Im Inneren der Zelle wurde die Calcium-Konzentration erhöht, dabei mit Hilfe der Patch-Clamp-Methode der Chlorid-Strom durch die Zellmembran gemessen. Wie erwartet, öffneten sich bei normalen Riechzellen die Chloridkanäle – dagegen gab es bei den Knock-out-Mäusen ohne Ano2 keine solche Reaktion auf erhöhtes Calci-um. Das beweist: Ano2 ist tatsächlich der gesuchte Chloridkanal.

Doch wie wirkt sich diese Signalverstärkung auf die Geruchs-wahrnehmung aus? Um das zu untersuchen, fertigte Balázs Pál zunächst Elektroolfaktogramme am Riechepithel der Mäuse an. Dabei wird die Riechschleimhaut aus den Tieren herauspräpa-riert und in einer kleinen Kammer in eine Nährlösung gelegt. Das Gewebe wird fortlaufend überspült und mit verschiedenen Aro-men beduftet. Zugleich setzt man eine feine Glaselektrode auf das Epithel und misst die elektrische Antwort. Beim Vergleich

Gwendolyn Billig

Der Gruppe von Thomas Jentsch gelang es, den lang ge-suchten Chloridkanal der Riechschleimhaut zu identifizie-ren. Die Überraschung dabei: Geruchswahrnehmungen entstehen anders, als es in den Lehrbüchern steht.

W I E W I R G E R Ü C H E W A H R N E H M E N

12 13

F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2

A N D R E W J . R . P L E S T E D

W H A T M A K E S T H A T G L U T A M A T E R E C E P T O R S O F A S T ?

W A S M A C H T D E N G L U T A M A T-R E Z E P T O R S O S C H N E L L ?

W H AT M A K E S T H AT G L U TA M AT E R E C E P T O R S O FA S T ? W A S M A C H T D E N G L U TA M AT- R E Z E P T O R S O S C H N E L L ?

Some nerve cells send out up to 1000 electrical signals per second for us to be able to function in our daily lives; and it is here that the glutamate receptor plays a key role. Andrew Plested and Anna Carbone have dismantled this receptor in order to understand how it functions – and in the process have discovered more about a kind of molecu-lar deep sleep.

Nothing works without it: the glutamate receptor is one of the central players in our nervous system and is therefore subject of research projects throughout the world. Better understanding of its function, and being able to manipulate it precisely, could be a step forward for medicine; for this receptor not only enables thoughts, sensations and reactions, but also probably plays a role in disorders such as stroke, Parkinson’s disease and epilepsy.

A certain type of glutamate receptor, the so-called AMPA receptor, is a specialist for rapid reactions and it is the receptor most com-monly encountered in our synapses. It can be activated up to 1000 times per second by the neurotransmitter glutamate, each time opening its channel through which cations flood in – producing a excitatory electrical signal. “Nerve impulses need to be rapid for us to be able to understand our environment and react to it,” ex-plains Andrew Plested. “A tennis player, for example, recognises within a fraction of a second where the ball is going and takes off in the right direction. And whenever we hear sounds, our eardrum vibrates hundreds or thousands of times per second – only with very rapid nerve impulses are we able to interpret them.”

To respond over and over again in a short space of time, the gluta-mate receptor must never get stuck in any single state. Imagine a pump with a hand-crank. After drawing a charge of water from the well, the handle must be returned to its resting position in order to pump again. To pump a decent amount of water in good time, this return must be smooth and easy; the same is true for the AMPA re-ceptor. However, such a rapid return to action is clearly not always

necessary and desirable, for another type of glutamate receptor, the kainate receptor, has a very similar structure, but its recovery after activation is 100-fold slower by comparison, as if the pump handle gets stuck. What exactly distinguishes the two receptors and what are the mechanisms by which a rapid and at the same time highly precise signal is possible in the first place? In order to find this out, Andrew Plested and his colleague Anna Carbone set out to dismantle the two receptor types into their component parts and, having put the receptors back together again, to check them over from top to bottom, so to speak.

The glutamate receptor consists of four subunits, whose peptide chains traverse the membrane and together form the ion channel. The remarkable thing is that each of these subunits consists of do-mains that are clearly distinguishable from each other; evolution has apparently used modules to construct the molecular machin-ery. Two such modules (S1 and S2) combine to form the binding site for the activating glutamate on the outside of the nerve cell. The binding domain looks like a clamshell; if the neurotransmitter binds in the gap between these domains, the clamshell snaps shut. This changes the conformation of the entire receptor, causing the ion channel to open.

In the area of the ion channel, AMPA and kainate receptors are al-most identical, which is why Andrew Plested suspected that the de-cisive difference between fast and slow receptors might be found in these “clamshells”. In order to prove this, Plested and Carbone used genetic engineering methods to swap the two halves of the binding domains between the two respective receptors. Finding the right places to make the cuts was not exactly child’s play: in similar previous experiments, the receptor had lost its function. “It was rather like a blind man being asked to hit a target with a bow and arrow – you need to have a bit of luck,” says Plested. But finally the researchers had two functioning chimeric receptors, whose reactions they were able to test using the patch-clamp technique. For this purpose, they activated one or only a few of the recep-tors in minute sections of the cell membrane with the aid of rapid pulses of glutamate and measured the electric current that then flowed through the membrane in the microsecond to millisecond range. Their first finding: the rapid AMPA receptor recovered very slowly as a result of the domain transferred from the kainate recep-tor, while conversely the binding domain transferred to the kainate receptor made it into a rapid one.

But how exactly does a receptor become rapid or slow? Like all receptors, glutamate receptors jump back and forth between dif-

ferent states. If glutamate binds, then most receptors enter the active state, at least briefly, and the ion channel opens. Alterna-tively, however, the receptor can switch into a state called “de-sensitisation” in which glutamate is bound, but the ion channel nevertheless remains closed. This state seems necessary so that the receptor reacts rapidly and sensitively, but the disadvantage is that it is insensitive to glutamate for some time after activation.

“A kind of timer is built into the receptor,” explains Plested. “Oth-erwise, the individual signals would be too long and would blend into each other.”

Andrew Plested now simulated the functioning of the molecular machine in the computer, while changing the reaction constants for the switch between the different states. He compared the result with his patch-clamp measurements under different experi-mental conditions. Through this comparison, it became clear that the speed could by no means be explained by faster or slower binding of glutamate, or by a more rapid opening of the ion chan-nel. The measured observations could only be explained if one assumes that there must be a further condition of the receptor: some of the desensitised receptors apparently fall into a kind of deep sleep, so-called “deep desensitisation”. The receptor can wake up from this deep sleep and return to the active, open con-dition, but only slowly, like a pump with a stiff handle. The rapid AMPA type largely avoids deep desensitisation, but the slower kainate type cannot.

In order to narrow down the area of the receptor that determines the speed of reaction, Plested and Carbone then swapped indi-vidual amino acids in the S2 domain. Just two such point muta-tions were sufficient to drastically slow the AMPA type. In contrast, a series of five mutations was necessary in order to get the kain-ate type up to full speed. None of these mutations impaired the binding of glutamate, but clearly had an effect on the ability of the modules to influence the conformation of the entire recep-tor. “The domains that bind glutamate are by no means the rigid clamshells that we once imagined,” says Andrew Plested. “Rather, at least in AMPA receptors, they are ‘wobbly’, which means that

they can switch between different conformations easily. We imag-ine that this property is important for AMPA receptor function.”

Deep desensitisation may serve as a kind of safety valve, in order to prevent an over-activation of nerve cells. Surprisingly, this evo-lutionary tuning also regulates the speed of reaction of the gluta-mate receptor. This molecular fine adjustment probably reflects the different roles of the receptor types: whereas the AMPA type is the workhorse, used everywhere in the brain, the kainate receptor is used only at some specialized connections. This does not mean that the slower reactions are less important, for glutamate recep-tors are also responsible for signals needed in learning processes to strengthen certain nerve connections and create memories. Another example of the receptor’s importance is shown by the fact that it is damaged by inherited mutations in people with mental disabilities.

In disorders such as epilepsy, medicines are already being used to block the glutamate receptor. For other indications, similar substances have proved unspecific and caused psychoses and hallucinations. “Our long-term aim is to investigate the role of the glutamate receptor in the living brain,” says Andrew Plested, whose group is also part of the excellence cluster “NeuroCure” at the Charité in Berlin Mitte. “This may also be of importance in the treatment of certain disorders such as a stroke, where a danger-ously large amount of glutamate is secreted over a short period.”

Ohne ihn geht gar nichts: Der Glutamat-Rezeptor ist einer der zentralen Akteure in unserem Nervensystem und daher weltweit Gegenstand vieler Forschungsprojekte. Könnte man seine Funk-

It can be activated up to 1000 times per second by the neurotransmitter glutamate, each time opening its channel through which cations flood in – producing

a excitatory electrical signal.

This does not mean that the slower reactions are less important, for glutamate receptors are also responsible for signals needed in learning processes to strengthen

certain nerve connections and create memories.

W A S M A C H T D E N G L U TA M AT- R E Z E P T O R

S O S C H N E L L ?

Bis zu 1000 elektrische Signale müssen manche Nerven-zellen pro Sekunde aussenden, damit wir uns in der Welt zurechtfinden können; von zentraler Bedeutung ist dabei der Glutamat-Rezeptor. Andrew Plested und Anna Carbo-ne haben den Rezeptor auseinandergenommen, um seine Funktionsweise zu verstehen – und mehr über eine Art molekularen Tiefschlaf herausgefunden.

Viktoria Klippenstein (front),

Anna Carbone

14 15

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2W H AT M A K E S T H AT G L U TA M AT E R E C E P T O R S O FA S T ? W A S M A C H T D E N G L U TA M AT- R E Z E P T O R S O S C H N E L L ?

tionsweise besser verstehen und präzise manipulieren, würde dies die Medizin einen großen Schritt voranbringen; denn der Rezep-tor ermöglicht nicht nur Gedanken, Sinneseindrücke und Reaktio-nen, sondern spielt vermutlich auch eine Rolle bei Störungen wie Schlaganfällen, Parkinson und Epilepsie.

Ein bestimmter Typ von Glutamatrezeptor, der sogenannte AMPA-Rezeptor, ist ein Spezialist für schnelle Reaktionen, und er ist der in unseren Synapsen am häufigsten anzutreffende Rezeptor. Bis zu tausendmal in einer Sekunde kann er durch den Neurotransmitter Glutamat aktiviert werden, jedes Mal öffnet er seinen Kanal aufs Neue, durch den Kationen einströmen – wodurch ein erregendes elektrisches Signal erzeugt wird. „Nervenimpulse müssen mitun-ter extrem schnell sein, damit wir unsere Umwelt verstehen und reagieren können“, erklärt Andrew Plested. „Ein Tennisspieler zum Beispiel erkennt im Bruchteil einer Sekunde wohin ein Ball fliegen wird und hechtet in die richtige Richtung. Und bei allen Tönen, die wir hören, schwingt unser Trommelfell hunderte oder tausen-de Male in einer Sekunde – nur mit sehr schnellen Nervenimpul-sen können wir so etwas interpretieren.“

Um wieder und wieder in kürzester Zeit reagieren zu können, darf der Glutamatrezeptor niemals in einem fixen Zustand verharren. Man kann sich das vorstellen wie eine handbetriebene Wasser-pumpe: Hat man eine Ladung Wasser aus einem Brunnen beför-dert, muss der Pumpenschwengel wieder in die ursprüngliche Stellung zurückkehren, damit man aufs Neue pumpen kann. Um rasch pumpen zu können, muss der Schwengel leicht und mühe-los zurückgleiten; das Gleiche gilt auch für den AMPA-Rezeptor.Immer ist die schnelle Einsatzbereitschaft aber offenbar gar nicht nötig und erwünscht, denn ein anderer Typ von Glutamatrezep-tor, der Kainat-Rezeptor, ist zwar sehr ähnlich aufgebaut, doch bei ihm verläuft die Rückkehr in den aktiven Zustand hundertfach lang-samer – so als ob der Pumpenschwengel sehr schwergängig ist und steckenbleibt. Was genau unterscheidet die beiden Rezep-toren, und durch welche Mechanismen ist ein schnelles und dabei

hochpräzises Signal überhaupt möglich? Um das herauszufinden, machten sich Andrew Plested und seine Kollegin Anna Carbone daran, die beiden Rezeptortypen in ihre Bausteine zu zerlegen und die daraus neu zusammengesetzten Rezeptoren quasi auf Herz und Nieren zu überprüfen.

Der Glutamatrezeptor besteht aus vier Untereinheiten, deren Pep-tidketten jeweils die Membran durchspannen und gemeinsam den Ionenkanal bilden. Bemerkenswert ist, dass jede dieser Unterein-heiten aus klar voneinander unterscheidbaren Domänen bestehen; die Evolution hat die molekulare Maschinerie offensichtlich aus Modulen zusammengesetzt. Zwei solcher Module (S1 und S2) bil-den auf der Außenseite der Nervenzelle gemeinsam die Bindungs-stelle für das aktivierende Glutamat. Diese Bindungsstelle ähnelt in ihrem Aussehen einer Muschel; bindet der Neurotransmitter in der Spalte zwischen den Domänen, dann klappt die Muschel zu. Dadurch ändert sich die Konformation des gesamten Rezeptors, wodurch der Ionenkanal geöffnet wird.

Im Bereich des Ionenkanals sind AMPA- und Kainat-Rezeptor nahezu identisch. Daher vermutete Andrew Plested den entschei-denden Unterschied zwischen schnellem und langsamem Rezep-tor in diesen „Muschelschalen.“ Um das nachzuweisen, versetzten Plested und Carbone mit Hilfe von gentechnischen Methoden die beiden Hälften der Bindungsstelle in den jeweils anderen Rezep-tor. Die richtigen Schnittstellen zwischen den Domänen zu finden, war dabei nicht gerade trivial: Bei ähnlichen früheren Versuchen hatte der Rezeptor seine Funktion verloren. „Es war ein bisschen so, als ob man blind einen Scherenschnitt ausführt – es gehört auch eine Portion Glück dazu“, sagt Plested.

Doch schließlich hatten die Forscher zwei funktionierende chimä-re Rezeptoren, deren Reaktionen sie mit Hilfe der Patch-Clamp-Technik testen konnten. Dabei aktivierten sie einen oder nur wenige der Rezeptoren in winzigen Ausschnitten der Zellmem-bran mit Hilfe von schnellen Pulsen von Glutamat und maßen den elektrischen Strom, der daraufhin im Mikro- bis Millisekun-denbereich durch die Membran floss. Die erste Erkenntnis: Tat-sächlich wurde der AMPA-Rezeptor durch die Bindungsdomäne des Kainat-Rezeptors langsamer, d.h. er kehrte nun nur langsam in den aktiven Zustand zurück. Dagegen war der Kainat-Rezeptor umgekehrt durch die vertauschte Bindungsdomäne zum schnel-len Rezeptor geworden.

Doch wie genau wird ein Rezeptor schnell oder langsam? Wie alle Rezeptoren springen Glutamatrezeptoren zwischen verschie-

denen Zuständen hin und her. Bindet Glutamat, dann werden die meisten Rezeptormoleküle aktiviert, der Ionenkanal öffnet sich zumindest kurzfristig. Alternativ kann der Rezeptor aber auch in einen Zustand wechseln, den man „Desensibilisierung“ nennt, in dem Glutamat zwar gebunden, der Ionenkanal aber trotzdem geschlossen ist. Dieser Zustand scheint notwendig zu sein, damit der Rezeptor schnell und empfindlich reagiert; der Nachteil ist allerdings, dass er nach einer Aktivierung für eine winzige Zeit-spanne unempfindlich gegenüber Glutamat wird. „In den Rezep-tor ist quasi ein Timer eingebaut“, erklärt Plested. „Ansonsten wären die einzelnen Signale zu lang und würden ineinander ver-schwimmen.“

Andrew Plested simulierte nun die Funktionsweise der moleku-laren Maschine im Computer und veränderte dabei die Reak-tionskonstanten für die Wechsel zwischen den verschiedenen Zuständen. Das Ergebnis verglich er mit seinen Patch-Clamp-Mes-sungen unter verschiedenen Versuchsbedingungen. Durch die-sen Vergleich wurde klar, dass sich das Tempo keineswegs durch ein rascheres Binden und Ablösen des Glutamats erklären lässt, oder durch ein rascheres Öffnen des Ionenkanals. Die gemesse-nen Beobachtungen ließen sich nur erklären, wenn man annimmt, dass es noch einen weiteren Zustand des Rezeptors geben muss: Ein Teil der desensibilisierten Rezeptoren verfällt offenbar in eine Art Tiefschlaf, die sogenannte „Tiefe Desensibilisierung“. Aus die-sem Tiefschlaf kann der Rezeptor wieder erwachen und in den akti-ven, geöffneten Zustand zurückkehren – allerdings nur langsam, wie eine Pumpe mit einem eingerosteten Schwengel. Der schnel-le AMPA-Typ vermeidet die Tiefe Desensibilisierung weitgehend, der langsameren Kainat-Typ kann das nicht.

Um den Bereich des Rezeptors, der über das Reaktionstempo bestimmt, noch genauer einzukreisen, tauschten Plested und Car-bone schließlich noch einzelne Aminosäuren in der S2-Domäne gegen andere aus. Schon zwei solcher Punktmutationen genüg-ten, um den AMPA-Typ drastisch zu verlangsamen. Dagegen

war eine Serie von fünf Mutationen nötig, um den Kainat-Typ auf Hochtouren zu bringen. Keine dieser Mutationen beeinträchtig-ten die Bindung von Glutamat, sondern wirkten sich offensichtlich auf die Fähigkeit der Module aus, die Konformation des gesam-ten Rezeptors zu beeinflussen. „Die Domänen, die das Glutamat binden, sind wohl keineswegs die starren Muschelschalen, als die man sie sich einmal vorgestellt hat“, sagt Andrew Plested. „Zumin-dest im AMPA-Rezeptor sind sie eher ‚wabbelig‘, können also leicht zwischen verschiedenen Konformationen wechseln. Nach unserer Vorstellung ist diese Eigenschaft wichtig für die Funktion des AMPA-Rezeptors.

Die „Tiefe Desensibilisierung“ könnte als eine Art Sicherheits-ventil dienen, um eine Überaktivierung der Nervenzellen zu ver-meiden. Überraschenderweise wird durch dieses evolutionäre Feintuning zugleich auch die Reaktionsgeschwindigkeit des Glu-tamatrezeptors reguliert. Vermutlich spiegelt diese molekulare Feinjustierung die unterschiedliche Funktion der Rezeptortypen wieder: Der AMPA-Typ ist das „Arbeitspferd“ unter den Rezep-toren, er wird überall im Gehirn eingesetzt; den Kainat-Rezeptor dagegen findet man nur bei einigen speziellen Verbindungen. Die langsameren Reaktionen könnten aber nicht minder bedeutsam sein, denn Glutamat-Rezeptoren sorgen durch ihre Signale auch dafür, dass bei Lernprozessen bestimmte Nervenverbindungen verstärkt werden und Erinnerungen entstehen. Seine Bedeutung zeigt sich zum Beispiel auch daran, dass der Rezeptor bei Men-schen mit geistiger Behinderung mitunter durch vererbte Muta-tionen beschädigt ist.

Bei Krankheiten wie Epilepsie werden bereits Medikamente einge-setzt, die den Glutamat-Rezeptor blockieren. Bei anderen Indikati-onen haben sich solche Substanzen allerdings als zu unspezifisch erwiesen; sie können Psychosen und Halluzinationen verursachen.

„Unser langfristiges Ziel ist es, die Rolle des Glutamat-Rezeptors im lebenden Gehirn zu untersuchen“, sagt Andrew Plested, des-sen Arbeitsgruppe auch Teil des Exzellenzcluster „NeuroCure“ an der Charité in Berlin-Mitte ist. „Das könnte auch bei der Behand-lung mancher Störungen von Bedeutung sein, wie zum Beispiel bei einem Schlaganfall: Hier wird in kurzer Zeit gefährlich viel Glu-tamat ausgeschüttet.“

Bis zu tausendmal in einer Sekunde kann er durch den Neurotransmitter Glutamat aktiviert werden,

jedes Mal öffnet er seinen Kanal aufs Neue, durch den Kationen einströmen – wodurch ein erregendes

elektrisches Signal erzeugt wird.

Die langsameren Reaktionen könnten aber nicht minder bedeutsam sein, denn Glutamat-Rezeptoren sorgen durch ihre Signale auch dafür, dass bei Lern-prozessen bestimmte Nervenverbindungen verstärkt

werden und Erinnerungen entstehen.

Carbone AL, Plested AJR (2012) Coupled control of desensitization and gating by the ligand binding domain of glutamate receptors. Neuron 74: 845-857.

Marcus Wietstruk

Most GluA2 ligand binding domain mutants

have little effect on recovery (green) but

the double mutant E713T Y768R (orange)

has very slow recovery from desensitization.

16 17

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2

spin condition than is the case for hydrogen; the resonance that is obtained with radio waves is correspondingly stronger.

In order to produce hyperpolarised xenon effectively and con-tinuously, Schröder’s group at the FMP established the LEIPNIX polariser, which stands for “Laser Enabled Increase of Polarisa-tion for Nuclei of Imprisoned Xenon”. The compact device uses a high-performance laser diode to produce approximately 30 litres of a gas mixture containing Xe-129 per hour. The spin polarisa-tion of the xenon atoms it contains amounts to around 25% – by comparison, it only amounts to around 0.001% in hydrogen nuclei in clinical MRI devices.

But how can xenon atoms be used to identify structures in liv-ing organisms? This is where a chemical trick comes into play – specially constructed organic molecules. They contain a kind of cage (cryptophan) into which the xenon atoms diffuse and can be bound. As a result of the changed chemical environment, the en-ergy levels of the spin states are displaced and thus the frequency of the emitted radio waves; this makes it possible to distinguish between free and captured Xe atoms. The cryptophan molecule can in turn be combined with tailor-made binding units. In rou-tine clinical practice, these might for example be antibodies that detect surface markers of cancer cells or arteriosclerotic plaques.

The next problem, however, is that relatively few xenon atoms ad-here to the structures searched for using this method. Until recent-ly, the signals measured were therefore too weak for the method to function under physiological conditions. Leif Schröder’s group therefore uses the so-called Hyper-CEST procedure in order to amplify the signals; CEST stands for “chemical exchange satura-tion transfer”. In this process, the radio waves are emitted for a few seconds, which cancels the polarisation of the xenon atoms captured in the biosensor. The xenon atoms in the surrounding solution remain unaffected by the radio waves, since their reso-nance is on a different wavelength. However, since they diffuse into the cryptophan cage for just a few milliseconds and then back

D I A G N O S I S W I T H X E N O N AT O M S D I A G N O S E M I T X E N O N - AT O M E N

A method for scanning patients and detecting specific molecules and cells relevant to disease – this is the vision on which the group led by Leif Schröder is working. With the aid of xenon biosensors, doctors may one day be able to gain new insights into the human body. Schröder has achieved a decisive breakthrough in the development of this revolutionary diagnostic procedure: Using optimised imaging techniques, the FMP researchers can now detect biosensors within 100 seconds with an accuracy for which a patient would have to keep still for 1100 years using existing methods.

The ability to look inside the body revolutionised medicine – many diseases or internal injuries are only identified by doctors today thanks to modern imaging diagnostics, by scanning the human body with radio waves or radioactive isotopes. However, this view is still restricted: Although it is possible to obtain excellent im-ages of different types of tissue and physiological processes in magnetic resonance imaging (MRI), it is barely possible to identify finer details such as cell types or metabolic products in small con-centrations. This is achieved more successfully by positron emis-sions tomography with the aid of radioactive isotopes, although the spatial resolution is lower here and the diagnosis involves exposure to ionizing radiation.

If one wants to detect diseases at the earliest possible stage, one needs a technique that combines the advantages of both meth-ods. The physicist Leif Schröder and his group are working on such a technique that may one day provide doctors with images in this kind of detail. As in MRI, Leif Schröder is harnessing the nuclear spin of atomic nuclei in a very high magnetic field, where they can start to resonate with radio waves of certain frequencies. In the standard MRI, one measures the radio waves emitted by polar-ised hydrogen atoms. Although they only emit weak signals, they are ubiquitous as a part of water in the body. The disadvantage: On the basis of the distribution of water molecules, it is virtually impossible to detect early biochemical changes that may lead to diseases.

Leif Schröder and his colleagues are therefore working with xenon atoms – even if this inert gas does not occur in the human body at all. Xenon has the advantage that the atoms can be brought into a hyperpolarised state with the aid of gaseous rubidium by means of spin coupling. Far more atoms are then available in a certain

out again, thousands of atoms are so to speak turned off when an image is taken, which leads to a dark spot in the image.

Until the final breakthrough was made, various technical improve-ments had to be made, such as a new technique of xenon “deliv-ery”. Whereas the unstable hyperpolarised xenon was previously collected in a cryogenic trap, Schröder and his colleagues can now produce and introduce it continuously – thanks to a particu-larly precise mass flow regulator, which ensures a constant gas flow. At the same time, the two postgraduate students Martin Kunth and Jörg Döpfert have succeeded in decisively improving the processing of the signals and thus the resolution of the images. Measurements are taken with the technically sophisticated “sin-gle-shot echo-planar imaging”(EPI) imaging sequence, in which the entire image information is determined in one go, instead of being measured stepwise as in older procedures.

The FMP researchers recently demonstrated the impressive capa-bilities of the technique with experiments in which they measured

the distribution of soluble biosensors in test tubes. They are now able to detect much lower concentrations of captured xenon atoms (30 nM), and that at a five-fold greater image resolution.

“We had to demonstrate that the method yields high-resolution images, and thus could in principle compete with the existing medical diagnostic procedures,” explains Schröder. “We now use the biosensors in concentrations that are realistic for practical application.” Whereas a measurement used to take over twenty minutes, now only 100 seconds are required. “Using conventional detection, one would need 1100 years to obtain such an image,” adds Jörg Döpfert.

Worldwide, just a few select groups are working on this cutting-edge technology of xenon biosensors. After Leif Schröder submit-ted his data to “Angewandte Chemie International Edition”, the journal immediately rated his work as a “Hot Topic”. In this paper, the FMP researchers also demonstrate that different xenon sen-sors can in principle be used simultaneously, sending out signals at different frequencies. This means that different disease markers could be identified in a patient with one scan and presented with different colours in the image. Thus, for example, it may be pos-sible to visualise the different cell types that make up a tumour.

It may even be possible to demonstrate the course of a process over time, as shown by the group in diffusion experiments. Again, this may be relevant to routine clinical practice where, for example, tumour cells often take up contrast medium more rapidly than healthy tissue.

The cryptophan molecule can in turn be combined with tailor-made binding units. In routine clinical practice, these might for example be antibodies that detect surface

markers of cancer cells or arteriosclerotic plaques.

“We had to demonstrate that the method yields high-resolution images, and thus could in principle

compete with the existing medical diagnostic procedures,” explains Schröder.

Martin Kunth, Jörg Döpfert and Dr. Leif Schröder

with a modell of the Xenon-cage

L E I F S C H R Ö D E R

D I A G N O S I S W I T H X E N O N A T O M S

D I A G N O S E M I T X E N O N - A T O M E N

18 19

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2

Der Blick ins Innere des Körpers hat die Medizin revolutioniert – viele Erkrankungen oder innere Verletzungen erkennen Ärzte heute nur dank moderner bildgebender Diagnostik, indem sie den menschlichen Körper mit Radiowellen oder radioaktiven Isotopen durchleuchten. Doch dieser Blick ist noch immer eingeschränkt: Im Kernspintomographen (MRT) kann man zwar hervorragend unter-schiedliche Gewebearten und physiologische Vorgänge sichtbar machen, aber kaum Feinheiten wie Zelltypen oder Stoffwechsel-produkte in geringer Konzentration erkennen. Das gelingt besser bei der Positronenemissions-Tomographie mit Hilfe radioaktiver Isotope, allerdings ist hier die räumliche Auflösung geringer und die Diagnose ist mit einer Strahlenbelastung verbunden.

Will man Erkrankungen im frühestmöglichen Stadium erkennen, dann braucht man ein Verfahren, das die Vorteile beider Methoden vereint. Der Physiker Leif Schröder arbeitet mit seiner Gruppe an einem solchen Verfahren, das Ärzten einmal entsprechend detail-lierte Bilder liefern könnte. Wie beim MRT nutzt auch Leif Schröder den Kernspin von Atomkernen in einem sehr hohen Magnetfeld, in dem sie mit Radiowellen bestimmter Frequenzen in Resonanz treten können. Beim herkömmlichen MRT misst man die von pola-risierten Wasserstoffatomen ausgesandten Radiowellen. Sie sen-den zwar nur schwache Signale aus, sind aber als Teil von Wasser im Körper allgegenwärtig. Der Nachteil dabei: Anhand der Vertei-

das Journal die Arbeit sogleich als „Hot Topic“ ein. In dem Paper demonstrieren die FMP-Forscher auch, dass man im Prinzip unter-schiedliche Xenon-Sensoren zugleich einsetzen kann, die bei ver-schiedenen Frequenzen Signale aussenden. So könnte man mit einer Aufnahme unterschiedliche Krankheits-Marker im Patienten auffinden und in einem Bild bunt darstellen. Damit könnte man zum Beispiel die unterschiedlichen Zelltypen sichtbar machen, aus denen sich ein Tumor zusammensetzt.

Sogar zeitliche Verläufe sind möglich, wie die Gruppe in Diffusi-onsexperimenten demonstriert hat. Auch das könnte im klinischen Alltag relevant werden, wo beispielsweise Tumorzellen häufig Kon-trastmittel schneller aufnehmen als gesundes Gewebe.

„Wir sind nun an dem Punkt angelangt, wo wir beginnen kön-nen, lebende Proben zu untersuchen“, sagt Schröder. Die Visi-on geht dahin, dass Patienten einmal das ungiftige Edelgas ein-atmen werden, so dass es sich zunächst in der Lunge und über das Blut im Körper verteilt. In einem Xenon-Kernspintomogra-phen der Zukunft könnte man dann im Patienten molekulare Veränderungen aufspüren und so vielleicht Krankheiten stop-pen, bevor sie überhaupt zu einem Problem werden.

D I A G N O S I S W I T H X E N O N AT O M S D I A G N O S E M I T X E N O N - AT O M E N

lung der Wassermoleküle lassen sich kaum frühzeitige biochemi-sche Veränderungen aufspüren, die zu Krankheiten führen könnten.

Leif Schröder und seine Kollegen arbeiten daher mit Xenon-Ato-men – auch, wenn das Edelgas im menschlichen Körper gar nicht vorkommt. Xenon hat den Vorteil, dass sich die Atome mit Hilfe von angeregtem gasförmigem Rubidium durch Spinkopplung in einen hyperpolarisierten Zustand versetzen lassen. Es liegen dann weit mehr Atome in einem bestimmten Spin-Zustand vor, als das bei Wasserstoff der Fall ist; entsprechend stärker ist das Signal, das man durch die Resonanz mit Radiowellen erhält.

Um effektiv und kontinuierlich hyperpolarisiertes Xenon zu erzeu-gen, hat die Gruppe um Schröder am FMP den LEIPNIX-Polari-sator etabliert, was für „Laser Enabled Increase of Polarization for Nuclei of Imprisoned Xenon“ steht. Das kompakte Gerät erzeugt mittels einer hochleistungsfähigen Laser-Diode pro Stunde ca. 30 Liter eines Gasgemisches, das Xe-129 enthält. Die Spin-Polarisati-on der Xenon-Atome darin beträgt etwa 25% – im Vergleich dazu beträgt sie bei Wasserstoffkernen in klinischen MRT-Geräten nur etwa 0,001%.

Doch wie kann man Xenon-Atome einsetzen, um Strukturen in lebenden Organismen zu erkennen? Hierfür gibt es einen che-mischen Trick – speziell konstruierte organische Moleküle. Sie enthalten eine Art Käfig (Cryptophan), in den die Xenon-Ato-me hinein diffundieren und gebunden werden können. Durch die veränderte chemische Umgebung verschieben sich die Ener-gieniveaus der Spinzustände und damit die Frequenz der ausge-sandten Radiowellen; man kann also zwischen freien und einge-fangenen Xe-Atomen unterscheiden. Die Cryptophan-Moleküle wiederum kann man mit maßgeschneiderten Detektoren verbin-den. Im klinischen Alltag könnten das zum Beispiel Antikörper sein, die Oberflächenmarker von Krebszellen oder Arteriosklero-se-Plaques erkennen.

Doch auch dann besteht noch das Problem, dass auf diese Weise nur vergleichsweise wenige Xenon-Atome an die gesuch-ten Strukturen angeheftet werden könnten. Bis vor kurzem waren die gemessenen Signale daher zu schwach, als dass das Verfah-ren unter physiologischen Bedingungen hätte funktionieren kön-nen. Die Gruppe von Leif Schröder verwendet daher das soge-nannte Hyper-CEST-Verfahren, um die Signale zu verstärken; CEST steht für „chemical exchange saturation transfer“. Dabei werden die Radiowellen einige Sekunden lang ausgesandt, wodurch die Polarisierung der im Biosensor gefangenen Xenon-Atome aus-gelöscht wird. Die Xenon-Atome in der umgebenen Lösung blei-ben davon unberührt, denn ihre Resonanz liegt auf einer anderen Wellenlänge. Da sie aber beständig für nur wenige Millisekunden in den Cryptophan-Käfig hinein- und wieder hinaus diffundieren, werden während einer Aufnahme Tausende Atome quasi ausge-knipst, wodurch ein dunkler Fleck im Bild entsteht.

Bis zum endgültigen Durchbruch waren aber noch einige techni-sche Verbesserungen notwendig, wie zum Beispiel eine neue Tech-nik der Xenon-„Anlieferung“. Während früher das instabile hyper-polarisierte Xenon in einer Kältefalle gesammelt wurde, können es Schröder und Kollegen nun kontinuierlich produzieren und einlei-ten – dank eines besonders präzisen Massenflussreglers, der den konstanten Gas-Strom sicherstellt. Zugleich ist es den beiden Dok-toranden Martin Kunth und Jörg Döpfert gelungen, die Verarbei-tung der Signale und damit die Auflösung der Bilder entscheidend zu verbessern. Gemessen wird mit der technisch anspruchsvollen

„single-shot echo-planar imaging“(EPI)-Bildgebungssequenz, bei der die gesamte Bildinformation auf einmal ermittelt wird, anstatt sie wie bei älteren Verfahren schrittweise zu vermessen.

Die Leistungsfähigkeit des Verfahrens haben die FMP-Forscher jüngst eindrucksvoll mit Versuchen unter Beweis gestellt, bei denen sie die Verteilung von löslichen Biosensoren in Teströhr-chen vermessen haben. Sie können nun deutlich geringere Kon-zentrationen gefangener Xenon-Atome (30 nM) darstellen, und das bei einer fünffach gesteigerten Bildauflösung. „Wir mussten beweisen, dass die Methode hochauflösende Bilder liefert, und damit im Prinzip mit den bisherigen medizinischen Diagnosever-fahren konkurrieren könnte“, erklärt Schröder. „Die Biosensoren setzen wir jetzt in Konzentrationen ein, wie sie für die Praxis rea-listisch sind.“ Während zuvor eine Messung über 20 Minuten dau-erte, sind jetzt nur noch 100 Sekunden nötig. „Bei konventioneller Detektion bräuchte man für eine solche Aufnahme 1100 Jahre“, erläutert Jörg Döpfert.

An der noch jungen Technologie der Xenon-Biosensoren arbei-ten weltweit nur einige wenige Gruppen. Nachdem Leif Schröder seine Daten bei „Angewandte Chemie“ eingereicht hatte, stufte

Die Cryptophan-Moleküle wiederum kann man mit maßge-schneiderten Detektoren verbinden. Im klinischen Alltag könn-ten das zum Beispiel Antikörper sein, die Oberflächenmarker

von Krebszellen oder Arteriosklerose-Plaques erkennen.

Patienten durchleuchten und dabei gezielt krankheitsre-levante Moleküle und Zellen aufspüren – an dieser Visi-on arbeitet die Gruppe von Leif Schröder. Mit Hilfe von Xenon-Biosensoren könnten Ärzte einmal ganz neue Ein-blicke in den menschlichen Körper gewinnen. Bei der Ent-wicklung des revolutionär neuen Diagnose-Verfahrens ist Schröder ein entscheidender Durchbruch gelungen: Durch optimierte Aufnahmetechniken können die FMP-Forscher nun Biosensoren innerhalb von 100 Sekunden mit einer Genauigkeit abbilden, für die ein Patient bei bisherigen Techniken 1100 Jahre stillhalten müsste.

“We have now reached the point at which we can start to investi-gate living samples,” says Schröder. The vision takes us to a day when patients will inhale the non-toxic inert gas so that it can be taken up in the lungs and distributed around the body in the bloodstream. In a xenon magnetic resonance scanner of the fu-ture, it would then be possible to detect molecular changes in the patient and thus maybe stop diseases before they ever become a problem.

D I A G N O S E M I T X E N O N - A T O M E N

„Wir mussten beweisen, dass die Methode hochauflösende Bilder liefert, und damit im Prinzip

mit den bisherigen medizinischen Diagnoseverfahren konkurrieren könnte“, erklärt Schröder.

Kunth M, Döpfert J, Witte C, Rossella F, Schröder L (2012) Optimized use of reversible binding for fast and selective NMR localization of caged xenon. Angew Chem Int Ed 51: 8217-8220. (hot paper, inside back cover article)

Model of a cryptophane cage containing capturing Xenon atoms

20 21

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2

V O L K E R H A U C K E

that looks a bit like a football. This lattice stabilises the invagina-tion of the membrane until finally a vesicle measuring around 100 nm pinches off. In this way, the cell is able to transport membrane proteins or molecules contained in the vesicle lumen inwards. However, clathrin-mediated endocytosis also has its dark sides. For example, many bacteria or viruses such as HIV use this gate-way in order to pass into the cell and multiply there.

But why does the spontaneous accumulation of clathrin occur in the first place? As fundamental as this process is for cellular function, surprisingly little was known about it. Many other acces-sory proteins are involved in this process, accumulating to form complexes at those points at which the clathrin legs come into contact with their terminal domains. It seemed most probable that these proteins mediate the binding of clathrin molecules to the membrane.

In order to find a substance that could stop vesicle transport, Volker Haucke, who was still researching at the Freie Universität Berlin in 2011, turned to Jens von Kries, who heads the Screening Unit at the FMP. “I needed someone who had lots of experience in the development of such assays, who was familiar with the complicated controls required and could also structure the initial findings according to chemical aspects. Jens was a massive help here.” In particular, Haucke was interested in finding small mol-ecules that specifically block the binding of clathrin to amphiphy-sin. As in most of the other accessory proteins, this binding takes place via an amazingly simple architecture: so-called clathrin-box motifs bind to the terminal domain of the clathrin molecules. If one managed to throw a spanner in the works at this point, one could interfere with the binding of numerous molecules to clathrin.

“It was a high-risk project with an uncertain outcome”, recollects Jens von Kries. “A pharmaceutical company would not attempt something like that.” With his help, a test procedure was devel-oped for high-throughput robots and, in the FMP’s substance library, researchers eventually found two candidates that specifi-cally blocked the binding of clathrin and amphiphysin. Through

B L O C K A D E I N T R A N S P O R T

B L O C K A D E I N T R A N S P O R T

B L O C K A D E I M T R A N S P O R T

B L O C K A D E I M T R A N S P O R T

Each cell has to take up and transport numerous substances. A central mechanism in this process involves minute vesicles that pinch off from the plasma membrane and pass into the cell. In co-operation with others, Volker Haucke’s group has developed small molecules with which this transport pathway can be specifically interrupted. They have thus not only explained the mechanism of vesicle formation – the new active substances may also form a basis for therapies of the future.

One might imagine the interior of a cell as being a gigantic airport: thousands of people come and go, meet up, or wander about through the crowd in apparent confusion. In the same way, pro-tein substances and other molecules diffuse through each cell, are sometimes transported inwards, then back out again, adhere to each other, pass on signals, and then separate again. Despite all advances, the possibilities for intervening with this process have been limited to date. “Forty percent of all medicines are directed against a certain type of receptor that emanates from the cell,” says FMP director Volker Haucke. Other active substances often target enzymes that are easy to inhibit. “The majority of cellular processes are poorly accessible,” according to Haucke. Up to now, the development of medicinal products has looked some-thing like a large-scale police search limited only to those people standing in front of the entrance to the airport or those whose fingerprints are already known.

In order to penetrate further into the cell, Volker Haucke set out to search for an active substance that would influence a central transport pathway into cells: clathrin-mediated endocytosis. This transport process begins with the attachment and assembly of clathrin molecules, causing the cell’s plasma membrane to invagi-nate and form a small pit. Each clathrin protein molecule has a triskelion shape, in other words three “legs” project outwards in a symmetrical pattern. At the end of each leg, the peptide chain folds and forms the globular terminal domain. Put together, the three-legged clathrin molecules form a spherical lattice structure

further chemical modification in collaboration with medicinal chemists from Australia, the researchers finally obtained two sub-stances, which they christened “pitstops”, one of which (pitstop 2) could also penetrate into cells. The pitstops were non-toxic for cells and x-ray structural analysis revealed that, as hoped, they compete with clathrin-box motifs of endogenous accessory pro-teins for the same binding site on the clathrin terminal domain, thus blocking it.

The next question, of course, was what effect the pitstops would have in living cells. Lisa von Kleist, then a postgraduate student in Hauckes group, used cell cultures to demonstrate that pitstop 2 disrupts the uptake of the iron transporter transferrin into cells, as well as that of epidermal growth factor, which is usually taken up together with its receptor by endocytosis. The penetration of HI viruses was also blocked by pitstops in cell culture, proof of the theory that the viruses pass into human cells via clathrin-mediated endocytosis.

When cell biologists get their hands on a new active substance, they have a tool at their disposal with which they can gain a bet-ter understanding of the processes that take place in cells. Since pitstops apparently stalled clathrin endocytosis, the researchers were now presented with the opportunity to examine this process in detail. To this end, Wiebke Stahlschmidt used cells in which the clathrin molecules fluoresce as a result of genetic modification, and observed the effect of pitstops under the TIRF microscope. TIRF stands for “total internal reflection fluorescence”; in this form of fluorescence microscopy, the incident light is concentrated on a small area by refraction in the lens, thus making it possible to observe the processes on cell membranes particularly well and in high resolution.

A surprising picture was revealed under the TIRF microscope. De-spite the pitstops, clathrin molecules continued to accumulate on the inside of the membrane, and vesicles pinched in. But while the whole process normally takes around one minute, the individual vesicles now remained where they were up to the end of the

microscopic imaging. In a snap-shot of the electron microscope, vesicles in all stages of the pinching-in process were found at the same time. “The formation of vesicles was so to speak frozen by the pitstops, only proceeding extremely slowly,” in the words of Volker Haucke.

Thus, it is now clear that clathrin can accumulate and form spheri-cal lattices, even in the absence of its direct binding to accessory proteins. It appears that the three-legged clathrin molecules with their terminal domains indeed specify the points at which the protein complexes adhere that are so important for the further dynamics and the course of endocytosis. Through further ex-periments with various fluorescing accessory proteins, Wiebke Stahlschmidt additionally showed the points of endocytosis at which the blockade sets in.

This also has consequences in nerve cells, where synaptic vesicle membranes have to be endocytosed. Vesicles are then loaded with neurotransmitters that are secreted into the synaptic cleft fol-lowing an electrical impulse. Haucke’s group showed that pitstops, injected into the giant axons of the nerve cells of river lampreys, interrupt stimulus conduction. It is possible that such a mechanism of action will one day be used to suppress the elevated activity of nerve cells in epilepsy.

In the meantime, research with the pitstops continues. Clathrin not only determines the structure for vesicle transport, the pro-tein building blocks also play an important role in cell division – the rigid protein molecules are a component of the spindle-shaped structures through which the chromosomes are pulled

“It was a high-risk project with an uncertain outcome”, recollects Jens von Kries. “A pharmaceutical company

would not attempt something like that.”

The penetration of HI viruses was also blocked by pitstops in cell culture, proof of the theory that the viruses

pass into human cells via clathrin-mediated endocytosis.

“With the pitstops we have identified a new mechanism of action that intervenes in fundamental

processes in the cell,” says Volker Haucke.

Confocal image of fixed cells with red antibody staining against clathrin.

The green channel shows EGF, the ligand for the EGF receptor. Nuclei are

stained with DAPI in blue.

22 23

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2

Das Innere von Zellen könnte man sich vorstellen wie einen gigan-tischen Flughafen: Tausende Menschen kommen und gehen, tref-fen aufeinander oder laufen scheinbar wirr durchs Gedränge. Ent-sprechend diffundieren durch jede Zelle Eiweißstoffe und andere Moleküle, werden mal hinein befördert, dann wieder hinaus trans-portiert, haften zusammen, geben Signale weiter und trennen sich wieder. Die Möglichkeiten, in dieses Geschehen einzugreifen, sind bei allen Fortschritten bislang nur begrenzt. „Vierzig Prozent aller

So lassen sich die Vorgänge an Zellmembranen besonders gut und hochaufgelöst betrachten. Im TIRF-Mikroskop bot sich ein über-raschendes Bild. Trotz der Pitstops lagerten sich weiterhin Cla-thrin-Moleküle an der Innenseite der Membran aneinander, und Vesikel schnürten sich ein. Doch während der ganze Prozess nor-malerweise etwa eine Minute dauert, blieben die einzelnen Vesi-kel nun bis zum Ende der mikroskopischen Aufnahme an Ort und Stelle stecken. In einer Momentaufnahme mit dem Elektronenmi-kroskop fanden sich zugleich Vesikel in allen Stadien der Einschnü-rung. „Die Bildung von Vesikeln war durch die Pitstops gleichsam eingefroren; sie lief nur noch stark verlangsamt ab“, beschreibt es Volker Haucke.

Damit ist nun klar, dass Clathrin auch ohne die angelagerten akzes-sorischen Proteine kugelförmige Gitter bilden kann. Es scheint, dass die dreibeinigen Clathrin-Moleküle mit ihren Terminal Domains überhaupt erst die Punkte vorgeben, an denen sich die Protein-komplexe anheften, die für die weitere Dynamik und den Verlauf der Endozytose so wichtig sind. Durch weitere Experimente mit verschiedenen, fluoreszierenden akzessorischen Proteinen konnte Wiebke Stahlschmidt zudem zeigen, an welchen Stellen der Endo-zytose die Blockade einsetzt.

Das hat auch in Nervenzellen Konsequenzen, denn hier müssen sich fortlaufend leere Vesikel einschnüren. Diese werden dann mit Neurotransmittern beladen, die im Fall eines elektrischen Impul-ses in den synaptischen Spalt ausgeschüttet werden. Die Gruppe von Haucke konnte zeigen, dass Pitstops, in die Riesenaxone der Nervenzellen von Flussneunaugen injiziert, die Erregungsweiter-leitung unterbrechen. Womöglich könnte man einen solchen Wirk-mechanismus einmal einsetzen, um bei Epilepsie die überhöhte Aktivität von Nervenzellen zu dämpfen.

Die Forschung mit den Pitstops geht indessen bereits weiter. Cla-thrin gibt nicht nur die Struktur für den Vesikeltransport vor, die Proteinbausteine spielen auch bei der Zellteilung eine wichtige Rolle – die starren Eiweißmoleküle sind ein Bestandteil der spin-delförmigen Strukturen, durch die Chromosomen bei der Teilung auseinandergezogen werden. Die mit Volker Haucke kooperie-renden Gruppen in Australien konnten inzwischen zeigen, dass man mit Pitstops die Teilung von Krebszellen stoppen kann, ohne gesunde Zellen zu beschädigen. Womöglich dient also Clath-rin auch einmal als Angriffspunkt für neuartige Krebstherapien.

„Mit den Pitstops haben wir einen neuen Wirkmechanismus auf-gezeigt, der in fundamentalen Vorgängen in der Zelle eingreift“, sagt Volker Haucke. „Möglich war das nur, weil wir einen span-nenden biologischen Prozess mit Hilfe von Werkzeugen der Che-mie untersucht haben.“

B L O C K A D E I N T R A N S P O R T B L O C K A D E I M T R A N S P O R T

Medikamente richten sich gegen eine bestimmte Art von Rezep-tor, der aus der Zelle herausragt“, sagt FMP-Direktor Volker Hau-cke. Andere Wirkstoffe zielen häufig auf Enzyme ab, die man leicht hemmen kann. „Der Großteil aller zellulären Prozesse ist schwer zugänglich“, so Haucke. Die Medikamentenentwicklung ähnelt bislang einer Großfahndung, die sich auf die Menschen beschränkt, die vor dem Eingang des Flughafens herumstehen oder deren Fingerabdrücke man schon kennt.

Um etwas weiter in die Zelle vorzudringen, machte sich Volker Hau-cke auf die Suche nach einem Wirkstoff, der einen zentralen Trans-portweg in Zellen, die von Clathrin vermittelte Endozytose, beein-flussen würde. Dieser Stofftransport beginnt damit, dass sich die Hüllmembran einer Zelle durch Anlagerung und Assemblierung von Clathrin-Molekülen nach innen einstülpt und ein kleines Grüb-chen bildet. Clathrin-Eiweißmoleküle sind jeweils wie ein Triskelion geformt, das heißt, im symmetrischen Abstand ragen drei „Beine“ starr nach außen. Am Ende jedes Beines faltet sich die Peptidket-te und formt die globuläre Terminal Domain. Aneinander gelegt bilden die dreibeinigen Clathrin-Moleküle eine kugelförmige Git-terstruktur, die an einen Fußball erinnert. Dieses Gitter stabilisiert die Einstülpung der Membran, bis sich schließlich ein etwa 100 nm großes Vesikel abschnürt. Auf diese Weise kann die Zelle Mem-branproteine oder auch im Vesikellumen befindliche Moleküle in ihr Inneres transportieren. Clathrin-vermittelte Endozytose hat aber auch ihre Schattenseiten. So benutzen viele Viren diese Pfor-te, um in das Zellinnere zu gelangen und sich dort zu vermehren.

Doch wie kommt es überhaupt zu der spontanen Zusammenlage-rung von Clathrin? So grundlegend dieser Prozess für die Zellfunk-tion ist, wusste man darüber überraschend wenig. Bei dem Prozess sind viele weitere akzessorische Proteine beteiligt, die sich an den Stellen zu Komplexen zusammenlagern, an denen die Clathrin-

Beine mit ihren Terminal Domains aufeinander stoßen. Am wahr-scheinlichsten schien, dass diese Proteine die Verbindung der Cla-thrin-Moleküle mit der Membran vermitteln.

Um eine Substanz zu finden, die den Vesikel-Transport stoppen könnte, wandte sich Volker Haucke, 2011 noch an der Freien Uni-versität Berlin beheimatet, an Jens von Kries, der die Screening Unit des FMP leitet. „Ich brauchte jemanden, der viel Erfahrung in der Entwicklung derartiger Assays hat, der sich mit den auf-wändigen, notwendigen Kontrollen auskennt und auch die ersten Funde nach chemischen Gesichtspunkten strukturieren kann. Jens war hier eine riesige Hilfe.“ Speziell ging es Haucke darum, klei-ne Moleküle zu finden, die spezifisch die Bindung von Clathrin an Amphiphysin blockieren. Wie bei den meisten anderen akzessori-schen Proteinen kommt diese Verbindung durch eine verblüffend einfache Architektur zustande: Sogenannte Clathrin-Box-Motive binden an die Terminal Domain der Clathrin-Moleküle. Könnte man an dieser Stelle Sand ins Getriebe streuen, so würde man die Verbindung von Clathrin mit einer Vielzahl von Molekülen stören.

„Es handelte sich um ein Hochrisiko-Projekt mit ungewissem Aus-gang“, erinnert sich Jens von Kries. „Ein Pharmaunternehmen würde so etwas nicht angehen.“ Mit seiner Hilfe entstand ein Test-verfahren für die Hochdurchsatzroboter, und schließlich fanden die Forscher in der Substanzbibliothek des FMPs zwei Kandida-ten, welche die Verbindung von Clathrin und Amphiphysin spe-zifisch blockierten. Durch weitere chemische Abwandlung, die in Kooperation mit Medizinalchemikern in Australien entstanden sind, erhielten die Forscher schließlich zwei Substanzen, die sie

„Pitstops“ (pit = englisch für Grube) tauften, und von denen eine (Pitstop 2) auch in Zellen eindringen konnte. Die Pitstops waren für Zellen ungiftig und in der Röntgenstrukturanalyse zeigte sich, dass sie wie erhofft mit Clathrin-Box-Motiven endogener akzesso-rischer Proteine um dieselbe Bindestelle an der Clathrin Terminal Domain konkurrierten, um diese so zu blockieren.

Natürlich war die nächste Frage, was die Pitstops in lebenden Zel-len bewirken würden. Lisa von Kleist, damals eine Doktorandin in Hauckes Gruppe, konnte anhand von Zellkulturen nachweisen, dass Pitstop 2 die Aufnahme des Eisen-Transporters Transferrin in Zellen unterbrach, ebenso die des Epidermalen Wachstums-faktors, der normalerweise zusammen mit seinem Rezeptor durch Endozytose aufgenommen wird. Auch das Eindringen von HI-Viren wurde in Zellkultur von den Pitstops blockiert, ein Beweis für die These, dass die Viren über die von Clathrin vermittelte Endozyto-se in menschliche Zellen gelangen.

Wenn Zellbiologen eine neue Wirksubstanz in Händen halten, dann besitzen sie damit auch ein Werkzeug, um die Vorgänge in Zellen besser verstehen zu lernen. Da Pitstops offensichtlich die Clathrin-vermittelte Endozytose unterbrachen, war nun die Gele-genheit gekommen, diesen Prozess genauer unter die Lupe zu nehmen. Für diesen Zweck benutzte Wiebke Stahlschmidt Zellen, in denen die Clathrin-Moleküle durch gentechnische Veränderun-gen fluoreszieren, und beobachtete die Wirkung von Pitstops unter dem TIRF-Mikroskop. TIRF steht für „total internal reflection“; bei dieser Form der Fluoreszenzmikroskopie wird der Lichteinfall durch Brechung am Objektiv auf einen kleinen Bereich beschränkt.

Jede Zelle muss eine Vielzahl von Stoffen aufnehmen und transportieren, ein zentraler Mechanismus hierfür sind winzige Vesikel, die sich von der Zellmembran in das Zell-innere abschnüren. In Zusammenarbeit mit anderen hat die Gruppe von Volker Haucke kleine Moleküle entwickelt, mit denen man diesen Transportweg gezielt unterbrechen kann. Damit haben sie nicht nur den Mechanismus der Vesikelbildung aufgeklärt – die neuen Wirkstoffe könnten auch zur Grundlage künftiger Therapien werden.

apart during division. The Australian groups cooperating with Volker Haucke have now shown that the cell division of cancer cells can be stopped with pitstops, without damaging healthy cells. It is thus possible that clathrin will also be a site of attack for novel cancer therapies one day.

“With the pitstops we have identified a new mechanism of action that intervenes in fundamental processes in the cell,” says Volker Haucke. “That was only possible, because we investigated a fas-cinating biological process with the aid of tools from the field of chemistry.”

B L O C K A D EI M T R A N S P O R T

„Es handelte sich um ein Hochrisiko-Projekt mit ungewissem Ausgang“, erinnert sich Jens von Kries. „Ein

Pharmaunternehmen würde so etwas nicht angehen.“

Auch das Eindringen von HI-Viren wurde in Zellkultur von den Pitstops blockiert, ein Beweis für die These,

dass die Viren über die von Clathrin vermittelte Endo- zytose in menschliche Zellen gelangen.

„Mit den Pitstops haben wir einen neuen Wirk-mechanismus aufgezeigt, der in fundamentalen Vorgängen

in der Zelle eingreift“, sagt Volker Haucke.

von Kleist L, Stahlschmidt W, Bulut H, Gromova K, Puchkov D, Robertson M, MacGregor KA, Tomlin N, Pechstein A, Chau N, Chircop M, Sakoff J, von Kries J, Saenger W, Kräusslich H-G, Shupliakov O, Robinson P, McCluskey A, Haucke V (2011) Role of the clathrin ter-minal domain in regulating coated pit dynamics revealed by small molecule inhibition. Cell 146: 471-484.

Custom-built laser combiner, to align 5 lasers of different

wavelengths for exciation of diverse fluorosphores into

the single molecule localization microscope.

Volker Haucke, Wiebke Stahlschmidt

24 25

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2

A L L H A N D S O N B O A R D : T H E C H E M I C A L B I O L O G Y U N I T

M I T V E R E I N T E N K R Ä F T E N : D I E C H E M I C A L B I O L O G Y U N I T

L E I B N I Z G R A D U AT E S C H O O L O F M O L E C U L A R B I O P H Y S I C S M I T V E R E I N T E N K R Ä F T E N : D I E C H E M I C A L B I O L O G Y U N I T

Chemical substances can influence the molecular machinery of life in manifold ways. For example, they can act as a medicine or as a poison, can be carcinogenic or protect against infection, or can be used diagnostically to detect diseases. Over 30 million different chemical substances are now available to scientists to work with, and new ones are constantly being added to the list – for chem-ists throughout the world are synthesising new compounds in their laboratories every day.

But how does one find the really interesting candidates among the vast range of different substances, and where in the living cells and organisms are the target structures and key switching points located at which new active substances can become effective? Over the past few years, optimum conditions have been created at the FMP to efficiently organise this search for the needle in the haystack. The “Screening Unit” headed by Jens Peter von Kries has been in existence at the institute since as far back as 2003. In order to extend support for research projects, an integrated tech-nology platform was created in which FMP scientists from different disciplines work hand-in-hand.

The Chemical Biology Unit comprises a total of eight modules (two modules in planning). They range from molecular drug de-sign, management of a large compound collection, process au-tomation, and the actual screening, up to cell biology and assay development, biophysical analysis, and database administration.

“The long-term future of the platform has now been secured,” to

the satisfaction of Ronald Frank, who together with Ronald Kühne and Jens Peter von Kries established the Chemical Biology Unit.

“The pivotal issue and exceptional value of this infrastructure is the initiation and support of collaborations between scientists within the institute, and with our cooperation partner MDC, as well as its openness to the scientific world. The Chemical Biology Unit of FMP will become a joint core facility of the recently founded Ber-lin Institute of Health (BIH). The unit’s concept opens up unique perspectives for collaboration with research groups from Europe and around the world, within the frame of research networks such as ChemBioNet and EU-OPENSCREEN, both initiated and coor-dinated by the FMP. On 29. April 2013, EU-OPENSCREEN was officially included into the German Roadmap for Large Research Infrastructures which also documents the willingness of the Minis-try to financially support the future European infrastructure and a significant upgrade of the Berlin site.”

The pharmaceutical chemist Bernd Rupp takes care of the infor-mation technology required to select suitable molecules from the massive, constantly growing range of substances available for the FMP collection as well as for further research projects.Under his management, a database with ultra-fast search routines was developed, which is being constantly improved. The chemist Michael Lisurek, who likewise belongs to the Drug Design group, aids Rupp to identify those molecules that have, based on their chemical and structural properties, the highest potential of being further developed and optimized (page 46). To this end, Lisurek calculates three-dimensional models of the molecules, which help to understand how the identified molecules might interact with their cellular target structures.

The chemist Edgar Specker organizes the management of the more than 60,000 substances now available on stock to the Screening Unit (page 118). “Our substances come from com-pletely different sources and from many different countries “, says Edgar Specker. ”When new compounds are to be added to the collection, it is also a matter of fascinating, novel chemistry; in

Over the past few years, a technology platform has been created at the FMP, to which research groups from the fields of chemical biology and structural biology have contributed and which has been developed into a com-prehensive overall concept. The Chemical Biology Unit extends the existing Screening Unit with the aim of more efficiently advancing the search for new active substances and biological probe molecules and further developing them in the field of medicinal chemistry.

The pivotal issue and exceptional value of this infrastructure is the initiation and support of collaborations between scientists within the institute, and with our cooperation

partner MDC, as well as its openness to the scientific world.

”When new compounds are to be added to the collection, it is also a matter of fascinating, novel chemistry;

in other words, such substances regularly differ much from substance classes already available.”

L E I B N I Z G R A D U A T E S C H O O L O F M O L E C U L A R

B I O P H Y S I C S

Die Förderung junger, talentierter Studenten und Wissenschaftler ist ein zentrales Anliegen am Leibniz-Institut für Molekulare Phar-makologie. Am Institut werden Bachelor-, Master-, Diplom- und Doktorarbeiten betreut.

In Zusammenarbeit mit anderen Instituten und Universitäten konzi-piert und organisiert das FMP ein Ausbildungsprogramm für Dok-toranden in molekularer Biophysik. Die Leibniz Graduate School of Molecular Biophysics nimmt 24 Berliner Doktoranden aus den Fächern Biologie, Chemie, Physik oder Medizin auf, die sich in ihrer Forschungsarbeit mit den Wechselwirkungen von Proteinen beschäftigen, also mit zentralen Vorgängen und Regelmechanis-men in Zellen und Organismen. Dabei kann es sich zum Beispiel um das Entstehen von viralen Infektionen handeln, oder auch um den Entstehungsprozess einer Alzheimer-Erkrankung. Entschei-dend für den Erfolg dieser Forschung ist eine große Bandbreite an biophysikalischen Techniken, wie zum Beispiel NMR-Spektro-skopie. „Um die Komplexität biologischer Systeme besser erfas-sen zu können, streben wir die Anwendung eines breiten Metho-denspektrums an“, erklärt der Koordinator Bernd Reif. Neben ihrer Forschungsarbeit besuchen die Doktoranden der Graduate School Praktika und Seminare und sollen damit ihr Wissen erwei-tern und andere Disziplinen kennenlernen. Auf diese Weise erhal-ten die jungen Wissenschaftler auch Gelegenheit, sich mit For-schern auf verwandten Gebieten zu vernetzen.

Die Leibniz Graduate School war im Gründungsjahr 2007 die erste Graduiertenschule der Leibniz-Gemeinschaft. Mittlerweile ist das Programm in der zweiten Förderperiode. Die Partnereinrichtun-gen sind: Technische Universität Berlin, Humboldt-Universität zu Berlin, Charité-Universitätsmedizin Berlin, Freie Universität Ber-lin, Universität Potsdam, Max-Delbrück-Centrum für Molekulare Medizin (MDC).

Promotion of young, talented students and scientists is a cen-tral concern of the Leibniz-Institut für Molekulare Pharmakologie. Bachelor’s, master‘s, diploma and doctoral theses are supervised at the Institute.

In cooperation with other institutes and universities, the FMP has conceived and organises a study programme for graduate students in molecular biophysics. The Leibniz Graduate School of Molecular Biophysics admits 24 doctoral students from Ber-lin from the subjects of biology, chemistry, physics or medicine, whose research work is centered around the interactions of pro-teins, i.e. with central processes and regulatory mechanisms in cells and organisms. For example, they may be researching into how viral infections occur, or into the processes behind the devel-opment of Alzheimer‘s disease. A decisive factor in the success of this research is a wide range of different biophysical techniques, such as NMR spectroscopy. „In order to better grasp the com-plexity of biological systems, we strive to apply a broad spectrum of methods,“ explains the coordinator Bernd Reif. Beside their re-search work, the students of the Graduate School attend practical training and seminars with the aim of extending their knowledge and getting to know other disciplines. In this way, the young sci-entists also have the opportunity to network with researchers in related fields.

In 2007, the year of its foundation, the Leibniz Graduate School was the first graduate school of the Leibniz-Gemeinschaft. In the meantime, the programme is in its second funding period. The partner institutions are: Technische Universität Berlin, Humboldt-Universität zu Berlin, Charité-Universitätsmedizin Berlin, Freie Uni-versität Berlin, Universität Potsdam, Max-Delbrück-Centrum für Molekulare Medizin (MDC).

26 27

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2

Chemische Substanzen können in vielfältiger Weise die moleku-lare Maschinerie des Lebens beeinflussen. Sie wirken beispiels-weise als Medikament oder als Gift, sind krebserregend oder schützen vor Infektionen oder können diagnostisch zum Aufspü-ren von Krankheiten genutzt werden. Über 30 Millionen verschie-dene chemische Substanzen stehen Wissenschaftlern inzwischen als Werkzeuge zur Verfügung, und ständig kommen neue hinzu – denn Chemiker aus aller Welt synthetisieren täglich neue Verbin-dungen in ihren Laboren.

Doch wie findet man in der unüberschaubaren Vielfalt der Stoffe die wirklich interessanten Kandidaten und wo sitzen in den leben-den Zellen und Organismen die Zielstrukturen und Schlüsselstel-len, an denen neue Wirkstoffe ansetzen können? In den vergan-genen Jahren wurden am FMP optimale Voraussetzungen dafür geschaffen, diese Suche nach der Nadel im Heuhaufen effizient zu gestalten. Bereits seit 2003 gibt es am Institut die „Screening Unit“ unter der Leitung von Jens Peter von Kries. Um die Unter-stützung von Forschungsprojekten zu erweitern, wurde eine inte-

A L L H A N D S O N B O A R D : T H E C H E M I C A L B I O L O G Y U N I T M I T V E R E I N T E N K R Ä F T E N : D I E C H E M I C A L B I O L O G Y U N I T

grierte Technologieplattform geschaffen, in der Wissenschaftler verschiedener Fachdisziplinen am FMP Hand in Hand arbeiten.

Insgesamt acht Module (zwei Module in der Planung) umfasst die Chemical Biology Unit. Sie reicht vom Wirkstoffdesign, dem Management der Substanzsammlung und der Prozessautomation über das eigentliche Screening bis hin zur Zellbiologie, der bio-physikalischen Analyse und der Datenbankverwaltung. „Die Platt-form ist nun langfristig abgesichert“, freut sich Ronald Frank, der zusammen mit Ronald Kühne und Jens Peter von Kries die Chemi-cal Biology Unit aufgebaut hat. „Das Entscheidende an dieser Inf-rastruktur ist die Vernetzung von Wissenschaftlern im Institut und mit unserem Kooperationspartner MDC sowie die Offenheit nach außen. Diese Struktur eröffnet Perspektiven zur Zusammenarbeit mit Arbeitsgruppen aus ganz Europa und der Welt im Rahmen von Netzwerken wie ChemBioNet und EU-OPENSCREEN, die beide durch das FMP initiiert und hier auch koordiniert werden.“ Am 29. April 2013 wurde EU-OPENSCREEN offiziell in die deutsche Roadmap für große Forschungsinfrastrukturen aufgenommen. Dadurch zeigt das zuständige Bundesministerium seine Bereit-schaft, die zukünftige europäische Infrastruktur und einen beacht-lichen Ausbau des Berliner Standortes finanziell zu unterstützen.

Um in dem riesigen, ständig wachsenden Angebot an Substanzen nach geeigneten Molekülen für die Sammlung des FMP und für weitere Forschungsaufgaben suchen zu können, ist der pharma-zeutische Chemiker Bernd Rupp für die Informationstechnologie zuständig. Unter seiner Leitung wurde eine Datenbank mit ultra-schnellen Suchmethoden entwickelt und ständig verbessert. Bei der Identifikation von Molekülen, die aufgrund ihrer chemischen und strukturellen Eigenschaften ausgewählt oder weiter verändert werden sollen, unterstützt ihn der Chemiker Michael Lisurek, der wie Rupp der AG Drug Design angehört (Seite 46). Er berechnet dreidimensionale Computerbilder der Moleküle, die helfen zu ver-stehen wie diese mit ihren zellulären Zielstrukturen wechselwirken.

Der Chemiker Edgar Specker koordiniert die mittlerweile mehr als 60.000 Substanzen, die der Screening-Unit zur Verfügung stehen

(Seite 118). „Unsere Substanzen kommen aus ganz unterschied-lichen Quellen und aus vielen verschiedenen Ländern“, erklärt Edgar Specker. „Bei Neuzugängen in der Probensammlung geht es auch um spannende Chemie, also um Stoffe, die sich möglichst von den schon vorhandenen Substanzklassen unterscheiden.“ Die Substanzen werden nicht einfach nur eingekauft: Edgar Spe-cker hat ein Portal geschaffen, über das Chemiker aus Deutsch-land und anderen europäischen Ländern ihre neu synthetisierten Substanzen in die Sammlung zur Charakterisierung und Nutzung einpflegen können.

Chemische Substanzen können in Lebewesen wirken, indem sie Zellrezeptoren aktivieren oder blockieren, Signalketten initiieren oder unterbrechen, oder Stoffwechselwege manipulieren. Um solche Wirkmechanismen zu entdecken, werden vom Team der Screening Unit um Jens von Kries biologische Testverfahren ent-wickelt. Sie werden dann von Robotern im Hochdurchsatz durch-geführt; mehr als 35.000 Tests können so an einem Tag in winzi-gen Volumina abgearbeitet werden. Dabei werden modernste Technologien eingesetzt: so können von Kries und seine Kolle-gen beispielsweise tausende Mikroskopaufnahmen von Zellen in Kultur mit spezieller Bildererkennungssoftware analysieren. Wich-tige Reagenzien wie spezielle synthetische Peptide werden dabei von der AG Peptidchemie zur Verfügung gestellt (Seite 102). Dafür, dass alle Roboter und Geräte miteinander kommunizieren, zusam-menarbeiten und die hochkomplexen Vorgänge des Hochdurch-satzscreenings (HTS) präzise und zuverlässig ausführen, sorgt der Biochemiker Martin Neuenschwander in der AG Screening Unit, der die Steuerung- und HTS-Datenanalyse-Programme entwickelt und anpasst. Alle Daten werden voll automatisiert erfasst, ausge-wertet und können dann für die Entscheidungsfindung dem Nut-zer der Plattform zur Verfügung gestellt werden.

Bei einem Wirkstoffscreening findet man immer eine ganze Reihe von interessanten Substanzen, sogenannten Hits, die aber selten bereits alle erwünschten Eigenschaften aufweisen. Um diese Sub-stanzen weiter zu optimieren, stehen Mitarbeiter des Moduls Syn-thesechemie in der AG Chemische Systembiologie bereit (Seite 110). Ab Mitte 2013 wird hier eine neue AG Medizinalchemie unter der Leitung von Marc Nazaré, einem erfahrenen Chemiker aus der Pharmaindustrie, ihre Arbeit zur Weiterentwicklung interessanter Hits zu Lead-Strukturen aufnehmen.

„Das Entscheidende an dieser Infrastruktur ist die Vernetzung von Wissenschaftlern im Institut und mit unserem Kooperationspartner MDC sowie die

Offenheit nach außen.“

„Bei Neuzugängen in der Probensammlung geht es auch um spannende Chemie, also um Stoffe, die sich möglichst von den schon vorhandenen Substanzklassen

unterscheiden.“

other words, such substances regularly differ much from sub-stance classes already available.” Those substances are not just being purchased: Edgar Specker has created a portal via which chemists from inside and outside Germany are invited to submit their newly synthesised substances to the collection for further characterisation and utilisation.

Chemical substances can have an effect in organisms by activating or blocking cell receptors, initiating or interrupting signal chains, or manipulating metabolic pathways. To discover compounds interfering with such mechanisms, the team of the Screening Unit led by Jens von Kries develops biological assays, test procedures, that are then performed by robots in high-throughput fashion; more than 35,000 assays can thus be processed in minute volumes on one day. Cutting-edge technologies are employed: for exam-ple, Kries and colleagues can analyse thousands of microscopic images of cells that have been treated with compounds in culture using special image recognition software. Furthermore, important reagents such as special synthetic peptides are made available by the Peptide Chemistry group (page 102).

Martin Neuenschwander, biochemist in the Screening Unit, assures that all of the robots and devices communicate with each other, work in concert and carry out the highly complex processes of the high-throughput screening (HTS) precisely and reliably. He devel-ops and adjusts the instrument controlling and HTS data analysis programs. All data are collected and analysed fully automatically before they are made available to the platform’s users for decision making.

The compound screening process always throws up a whole range of interesting substances, so-called hits, but they rarely show right from the beginning all of the properties desired. These substanc-es are further chemically modified and optimised by staff from the Synthesis Chemistry module of the Chemical System Biology group (page 110). From the middle of 2013, the new research group Medicinal Chemistry, headed by Marc Nazaré, an expe-rienced chemist from the pharmaceutical industry, will start its work aiming at the development of promising hits into qualified, so-called lead compounds.

In den letzten Jahren ist am FMP eine Technologieplatt-form entstanden, zu der Arbeitsgruppen aus den Berei-chen Chemische Biologie und Strukturbiologie beitragen und die zu einem umfassenden Gesamtkonzept ergänzt wurde. Die Chemical Biology Unit erweitert die bisherige Screening Unit, um die Suche nach neuen Wirkstoffen und biologischen Sondenmolekülen noch effizienter vo-ranzutreiben und diese medizinalchemisch weiterzuent-wickeln.

M I T V E R E I N T E N K R Ä F T E N :D I E C H E M I C A L B I O L O G Y

U N I T

C H E M I N F O R M AT I C S /C O M P U TAT I O N A L D R U G D E S I G N

Michael Lisurek

S Y N T H E T I C C H E M I S T RY

Ronald Frank

C O M P O U N DM A N A G E M E N T

Edgar Specker

A S S AY-D E V E L O P M E N T /C E L L B I O L O G Y

Jens Peter von Kries

P R O C E S S -A U T O M AT I O N

Martin Neuenschwander

B I O P H Y S I C A L P R O F I L I N G

Edgar Specker

S C R E E N I N G

Jens Peter von Kries

D ATA B A S ED E V E L O P M E N T /I T

Bernd Rupp

29

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2

28

N M R F O R T H E W H O L E O F E U R O P E N M R F Ü R G A N Z E U R O PA

Like few other technologies, NMR spectroscopy enables us to look into the very heart of matter, creating close-ups in atomic resolution. In a powerful magnetic field, some atomic nuclei con-tained in the samples behave like small magnets and orient them-selves to the outer field. Depending on the chemical environment, they can absorb the energy of radio waves, from which it is pos-sible to determine the structure of biological molecules, using complicated methods of calculation. Since this technology is very complex with its enormous supraconducting magnets, it requires considerable expertise; hence, it makes sense to open up the leading centres in Europe to biologists from other countries, in order to take a joint approach to solving particularly interesting research questions.

The European infrastructure project “Bio-NMR”, which was launched in September 2010 and in which the FMP is also partici-pating, is based on this idea. “The EU wants to make the large devices accessible to more scientists,” states Hartmut Oschkinat, head of the Section Structural Biology. “We receive money for making 20 percent of our measuring time available. In turn, we can then channel the European funding into investments – that makes it a win-win situation.” In particular, the FMP provides access to the solid-state NMR, which allows measurements on very complex samples, but also to the unique in-cell NMR of Philipp Selenko’s research group, in which samples are labelled with isotopes with-in living cells. Interested scientists must submit an application, but are supported by NMR experts at the FMP. However, a condition for the EU funding is that the scientists who apply belong to groups established outside of Germany.

Therefore, scientists from Spain, Sweden, France and England have visited the FMP over the past few years. They include Heike

Wie wenig andere Technologien ermöglicht die NMR-Spektro-skopie einen Blick ins Innerste der Materie, schafft Nahaufnah-men in atomarer Auflösung. In einem starken Magnetfeld werden manche Atomkerne der Proben selbst zu kleinen Magneten und richten sich entsprechend dem äußeren Feld aus. Je nach che-mischer Umgebung absorbieren sie dann die Energie von Radio-wellen, woraus sich in komplizierten Rechenverfahren die Struk-

Knicker from IRNAS-CSIC in Seville, Spain. The biochemist brought samples with her that have never before been investi-gated at the FMP: she analysed various soil types in order to solve the puzzling question of what happens to nitrogen compounds that are formed when plant residues decompose. “Soils are very complex in their composition and are not at all easy to investigate. Using standard methods, we can only identify 40 percent of the nitrogen compounds. I put forward the hypothesis that humus, in other words the organic soil material of our fields and gardens, is made up to 30 percent of peptides and thus plays a major role in carbon storage in the soil. That would be surprising, because pep-tides are generally considered to be ‘chocolate’ for microorgan-isms.” With the aid of the DNP solid-state NMR, Heike Knicker has now collected her first data at the FMP. She is convinced of this approach, is planning a first publication and would like to come back as soon as possible: “The application was uncomplicated, I was well looked after and have learnt a lot. The specialist knowl-edge at the Institute is truly impressive!”

Support for such individual projects is just one pillar of Bio-NMR. “One very attractive component of the project is the opportunity to collaborate with other leading groups,” says Hartmut Oschki-nat. “At the regular meetings, measuring techniques are dis-cussed, for example, and we have already jointly developed pro-grams for improving the algorithms used in evaluating data.”

tur biologischer Moleküle ermitteln lässt. Da die Technologie mit ihren riesigen supraleitenden Magneten sehr aufwändig ist und viel Expertise erfordert, liegt der Gedanke nahe, die in Europa füh-renden Zentren für Biologen aus anderen Ländern zu öffnen, um besonders spannende Forschungsfragen gemeinsam anzugehen.

Auf dieser Idee basiert das europäische Infrastrukturprojekt „Bio-NMR“, das im September 2010 ins Leben gerufen wurde und dem auch das FMP angehört. „Die EU möchte erreichen, dass mehr Wissenschaftler Zugang zu den großen Geräten bekom-men“, erklärt Hartmut Oschkinat, Leiter des Bereichs Strukturbio-logie. „Wir erhalten Gelder dafür, dass wir bis zu 20 Prozent unse-rer Messzeit zur Verfügung stellen. Die europäischen Fördermittel können wir dann wiederum in Investitionen stecken – es ist also eine Win-Win-Situation.“ Das FMP stellt insbesondere die Fest-körper-NMR zur Verfügung, die Messungen an sehr komplexen Proben erlaubt, wie auch die weltweit einzigartige In-Cell-NMR der Arbeitsgruppe von Philipp Selenko, bei der Proben innerhalb lebender Zellen mit Isotopen markiert werden. Die Interessenten müssen einen Antrag stellen, werden dabei aber von den NMR-Experten am FMP unterstützt. Bedingung für die EU-Förderung ist allerdings, dass antragsstellende Wissenschaftler Gruppen ange-hören, die außerhalb von Deutschland angesiedelt sind.

In den letzten Jahren haben daher Wissenschaftler aus Spanien, Schweden, Frankreich und England das FMP besucht. Unter ihnen auch Heike Knicker vom IRNAS-CSIC im spanischen Sevilla. Die Biochemikerin brachte Proben mit, wie sie am FMP wohl noch nie zuvor untersucht wurden: Sie analysierte verschiedene Bodensor-ten, um dem rätselhaften Verbleib der Stickstoff-Verbindungen, die beim Verrotten von Pflanzenresten entstehen, auf die Spur zu kommen. „Böden sind in ihrer Zusammensetzung sehr komplex und gar nicht so einfach zu untersuchen. Mit gängigen Metho-den können wir nur 40 Prozent der Stickstoffverbindungen identi-fizieren. Ich habe die Hypothese aufgestellt, dass Humus, also das organische Bodenmaterial unserer Äcker und Gärten, zu 30 Pro-zent aus Peptiden besteht und damit wesentlich an der Kohlen-stoffspeicherung im Boden beteiligt ist. Das wäre überraschend, weil Peptide allgemein als ‚Schokolade‘ für Mikroorganismen gel-ten.“ Mit Hilfe der DNP-Festkörper-NMR hat Heike Knicker nun am FMP erste Daten gesammelt. Sie ist überzeugt von diesem Ansatz, plant eine erste Veröffentlichung und möchte möglichst bald wieder kommen: „Der Antrag war unkompliziert, ich wurde gut betreut und habe viel gelernt. Das Fachwissen am Institut ist wirklich beeindruckend!“ Die Förderung solcher Einzelprojekte ist dabei nur eine Säule von

„Bio-NMR“. „Eine sehr attraktive Komponente des Projekts ist die Möglichkeit, mit anderen führenden Gruppen zu kooperieren“, sagt Hartmut Oschkinat. „Bei den regelmäßigen Treffen werden zum Beispiel neue Messtechniken diskutiert, und wir haben bereits gemeinsam Programme entwickelt, um die verwendeten Algorith-men bei der Auswertung zu verbessern.“

www.bio-nmr.net

„The EU wants to make the large devices accessible to more scientists,“

states Hartmut Oschkinat„Die EU möchte erreichen, dass mehr

Wissenschaftler Zugang zu den großen Geräten bekommen“, erklärt Hartmut Oschkinat.

Ümit Akbey,

Sascha Lange,

Barth-Jan van Rossum

NMR 2 Building NMR 1 Building

N M R F O R T H E W H O L E O F E U R O P E

N M R F Ü R G A N Z E U R O PA

Complex technology should be available to all scientists who have good ideas. The EU project “Bio-NMR” is there-fore supporting researchers from throughout Europe, who will now be given access to the NMR devices at the FMP.

Aufwändige Technologie sollte für alle Wissenschaftler mit guten Ideen verfügbar sein. Das EU-Projekt „Bio-NMR“ unterstützt daher Forscher aus ganz Europa, die auf diese Weise Zugang zu den NMR-Geräten am FMP erhalten.

N M R F Ü R G A N Z E U R O PA

R E S E A R C H H I G H L I G H T S / / / N M R F Ü R G A N Z E U R O PA

30 31

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2

Computational Chemistry / Drug DesignWirkstoff-Design

GROUP LEADERDr. Ronald Kühne

PAGE 46

Solution NMRLösungs-NMR

GROUP LEADERDr. Peter Schmieder

PAGE 50

Molecular ImagingMolekulare Bildgebung

GROUP LEADERDr. Leif Schröder

PAGE 54

In-Cell NMRNMR in Zellen

GROUP LEADERDr. Philipp Selenko

PAGE 58

NMR-supported Structural BiologyNMR-unterstützte Strukturbiologie

GROUP LEADERProf. Dr. Hartmut Oschkinat

PAGE 34

Protein Engineering

GROUP LEADERProf. Dr. Christian Freund

PAGE 38

Structural Bioinformatics and Protein DesignStruktur-orientierte Bioinformatik und Proteindesign

GROUP LEADERDr. Gerd Krause

PAGE 42

STRUCTURAL BIOLOGY SECTION

B E R E I C HS T R U K T U R B I O L O G I E

32 33

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2S T R U C T U R A L B I O L O G Y S E C T I O N B E R E I C H S T R U K T U R B I O L O G I E

stand protein function, especially of G-protein coupled receptors (GPCRs) via pharmacological interference. The “Drug Design” group led by Ronald Kühne accomplished a very demanding task in developing a set of small-molecule fragments that in-hibit protein-protein interactions involving proline-rich motifs, yielding efficient inhibitors of EVH1 and WW protein domain interactions. The “Structural Bioinformatics” group headed by Gerd Krause generated a new web-based system that assists in evaluating the molecular effects of genetic variations in GPCRs. The structure-function relationship of two receptors, LHR and TSHR, were deciphered to derive molecular details of activa-tion and inactivation patterns, and to understand the activity of agonists and antagonists. Both computational groups test the compounds developed in their biological laboratories, thereby closing the loop between structural studies and computational modelling, as well as biochemical and cell physiological analyses.

During the reporting period, Christian Freund, head of the FMP’s “Protein Engineering” group accepted a call as tenured professor (W2). In 2011, he transferred his group to the Freie Universität Berlin. Already in 2010, Bernd Reif and his “Solid-State NMR” group left for the Technische Universität München but kept his close ties with the FMP as demonstrated by a number of publi-cations during the reporting period. As the process of filling this vacant faculty position has been determinedly pursued, appoint-ing an excellent successor is expected for 2013. With its special combination of techniques (solution and solid-state NMR, In-cell technologies, MRI), the structural biology section is well suited to determine the molecular structures of cellular targets relevant for molecular pharmacology, the institute’s research mission.

Die Nachwuchsgruppe „Molekulare Bildgebung” von Leif Schrö-der geht ebenfalls neue Wege; sie entwickelt eine auf Biosenso-ren basierende, extrem sensitive Technik der NMR-Bildgebung, bei dem zur Verbesserung des Signal-Rausch-Verhältnisses Hyper-polarisation angewendet wird. Leif Schröder nutzt Magnetre-sonanztomographie (MRT) als Haupttechnik für physiologische Untersuchungen. Durch die Entwicklung einer hocheffizienten Technik für die Hyperpolarisation von Xenon, das dann zur Dar-stellung membrangebundener Sonden genutzt wird, sind für die MRT nur noch extrem geringe Stoffmengen zur Erzeugung eines gewebs- oder molekülspezifischen Kontrastes erforderlich. So könnten bald nicht-invasiv z.B. Krebserkrankungen in einem frü-hen Stadium nachzuweisen sein.

Die Cheminformatik-/Bioinformatikgruppen nutzen Strukturinfor-mationen zur Konstruktion von Hemmstoffen für Proteinwechsel-wirkungen. Gleichermaßen versuchen sie die Funktionsweise von Proteinen, speziell G-Protein-gekoppelter-Rezeptoren (GPCR), auf der Basis pharmakologischer Interferenz zu verstehen. Die Arbeitsgruppe „Wirkstoff-Design” von Ronald Kühne war sehr erfolgreich mit der Entwicklung einer Reihe niedermolekularer, prolinreicher Fragmente zu Hemmstoffen von Protein-Protein-Wechselwirkungen. Diese inhibieren äußerst effizient die Wech-selwirkung zwischen der EVH1- und der WW-Proteindomäne.

Die von Gerd Krause geleitete Arbeitsgruppe „Struktur-orientier-te Bioinformatik und Proteindesign” entwickelte ein neues Web-basiertes System, das die Analyse molekularer Auswirkungen genetischer Variationen in GPCRs unterstützt. Für Glykoprotein-hormonrezeptoren, zu denen auch der Luteinisierende-Hormon Rezeptor (LHR) und der Schilddrüsenstimulierende Hormonre-zeptor (TSHR) gehören, wurden die Struktur-Funktionsbeziehun-gen aufgeklärt. Daraus konnten detailliert molekulare Muster zur Rezeptoraktivierung und -deaktivierung abgeleitet und die Aktivi-tät von Agonisten und Antagonisten verstanden werden.

Beide Informatik-Arbeitsgruppen haben mittlerweile biologische Labore eingerichtet, um die von ihnen entwickelten Substanzen biologisch/biochemisch zu testen. Damit schließt sich der Kreis, bestehend aus strukturellen Untersuchungen, computergestütz-tem Modeling und biochemischen- und zellphysiologischen Ana-lysen.

Während des Berichtszeitraums wechselte Christian Freund, Leiter der Arbeitsgruppe „Protein Engineering”, auf eine Professur (W2) an der Freien Universität Berlin. Bereits 2010 ging Bernd Reif mit seiner Arbeitsgruppe „Festkörper-NMR” an die Technische Uni-versität München, hielt aber weiterhin engen Kontakt zum FMP, woraus eine Reihe gemeinsamer Publikationen resultierte. Die Neubesetzung der freigewordenen Stelle von Bernd Reif wurde zielstrebig verfolgt, sodass mit der Ernennung eines ausgezeich-neten Nachfolgers für 2013 zu rechnen ist. Der Bereich „Struk-turbiologie“ wird somit wieder das vollständige Spektrum an Forschungskompetenz auf dem Gebiet der NMR-basierten Struk-turbiologie vorweisen und damit bestens für die wissenschaftliche Mission des Instituts, die Bestimmung relevanter Zielstrukturen für Wirkstoffe, gerüstet sein.

Molecular pharmacology requires three-dimensional represen-tations of super-molecular arrangements within the cell, which are controlled in vivo by temporal and spatial coordination of protein expression, degradation, and post-translational modi-fication. The dynamic nature of these phenomena challenges static structure determination techniques and implies a strong incentive to employ Nuclear Magnetic Resonance spectroscopy (NMR). The department of “NMR-supported Structural Biology” develops and applies solution and solid-state NMR techniques to investigate proteins in native environments. Furthermore, mo-lecular modelling is applied to derive atomic-resolution struc-tural data that are indispensable on the path to pharmacological interference. With regards to the design of bio-active molecules, the development of protein interaction inhibitors is a major theme. Collaborations involving groups of the molecular medi-cine and chemical biology sections provide essential access to biological experiments or new chemical compounds, while ex-pertise in structural biology contributes to projects in the mo-lecular medicine department.

During the reporting period three-dimensional structural mod-els of alphaB crystallin oligomers and of the infection-relevant membrane protein YadA, an adhesin from Yersinia enterocolitica, were determined in the group “NMR-supported Structural Biolo-gy”, using solid-state NMR, by the team around Hartmut Oschkinat and Barth van Rossum. Furthermore, the application of microwave-based hyperpolarisation (DNP) yielded a picture of the nascent polypeptide chain growing inside the ribosome. The “Solution NMR” group of Peter Schmieder studied the transmission of light-induced signals – and the accompanying structural changes – in the blue light receptor YtvA. In addition, the connection between protein dynamics in MHC complexes and auto-inflammatory dis-orders is being investigated.

The “In-cell NMR” junior group headed by Philipp Selenko ex-plores novel NMR-based methodologies that allow monitoring of proteins inside cells. Projects include profiling of kinase activi-ties via peptide-based Kinase Activity Reporters (KARs). These allow measuring dynamic changes of cellular kinase activities under different physiological and pathophysiological conditions. Investigation of the structure of alpha-synuclein within cells aims at a molecular understanding of Parkinson’s disease.

The “Molecular Imaging” junior group of Leif Schröder works at the physiological level using MRI as a major technique and ex-plores new ground in developing a biosensor-based NMR-imag-ing approach, employing hyperpolarisation to enhance the signal-to-noise ratio. In the course of this work, Leif Schröder and his co-workers devised a highly efficient technique for hyper- polarizing xenon, which is then used to polarise membrane- embedded probes. MRI enhanced by hyperpolarisation enables the generation of tissue- or molecule-specific contrast and may be used to non-invasively detect, for example, cancer at its earliest stages.

The cheminformatics/bioinformatics groups utilize structural in-formation to derive protein interaction inhibitors, and to under-

Molekulare Pharmakologie benötigt die Kenntnis der dreidimen-sionalen Struktur von molekularen Komplexen. Solche Komplexe aus verschiedenen Proteinen und anderen Molekülen bilden sich und zerfallen kontinuierlich in Prozessen, die in lebenden Zellen u.a. durch die räumliche und zeitliche Kontrolle von Proteinex-pression, -abbau und chemische Modifikationen kontrolliert wer-den. Die moderne Strukturbiologie erforscht diese naturbedingt dynamischen Prozesse mit eigens dafür entwickelten Techniken der Kernspinresonanzspektroskopie (nuclear magnetic resonance, kurz NMR). Die Wissenschaftler der Abteilung „NMR-unterstütz-te Strukturbiologie“ nutzen und entwickeln Lösungs- und Fest-körper-NMR-Techniken, um Proteine in voller Länge und in ihrer natürlichen Umgebung zu untersuchen. Dazu gehört auch die molekulare Modellierung, da die Aufklärung der Struktur bio-logischer Zielmoleküle (Targets) mit einer Auflösung einzelner Atome unabdingbar ist, um in einem nächsten Schritt einen Weg zu deren pharmakologischer Beeinflussung zu finden.

Ein zentrales Forschungsthema am FMP ist die Hemmung von Proteinwechselwirkungen, basierend auf dem Design bioakti-ver Moleküle. Hier ist die Zusammenarbeit zwischen den Grup-pen und Bereichen essentiell: Gruppen der Bereiche „Moleku-lare Physiologie und Zellbiologie“ sowie „Chemische Biologie“ ermöglichen einen Zugang zu biologischen Experimenten und zu neuartigen chemischen Substanzen. Die Strukturaufklärung trägt wiederum entscheidend zu Projekten der Bereiche „Mole-kulare Physiologie und Zellbiologie“ und „Chemische Biologie“ bei.

Im Berichtszeitraum konnte das Team um Hartmut Oschkinat und Barth van Rossum mittels Festkörper-NMR die dreidimen-sionale Struktur der polydispersen AlphaB-Crystallin-Oligomere sowie des Membranproteins YadA bestimmen. Das Protein YaDA ist ein Adhäsin des Bakteriums Yersinia enterocolitica, ein krank-heitserregendes Bakterium, das Fieber und Durchfall auslöst. Wissenschaftlern des Teams gelang es ferner mittels mikrowel-lenbasierter Hyperpolarisation (DNP) ein Bild einer im Ribosom wachsenden Polypeptidkette während der Proteinbiosynthese zu gewinnen. Die Arbeitsgruppe „Lösungs-NMR“ (Peter Schmieder) untersuchte die Weiterleitung von Lichtsignalen und der damit verbundenen Strukturveränderungen in einem Blaulichtrezep-tor aus Bacillus subtilis. Des Weiteren wird derzeit die Beziehung zwischen der Proteindynamik in Multiproteinkomplexen, die für die Funktion des Immunsystems und bei autoinflammatorischen Erkrankungen eine wichtige Rolle spielen, untersucht.

Die von Philipp Selenko geleitete Nachwuchsgruppe „NMR in Zellen” geht neue Wege in der Entwicklung und Anwendung neuartiger NMR-Methoden, die die Beobachtung von Proteinen in lebenden Zellen erlauben. Eines der Projekte ist z.B. das Profi-ling von Kinase-Aktivitäten über peptidbasierte Kinase-Aktivitäts-Reporter (KAR). Diese ermöglichen die Messung dynamischer Änderungen der zellulären Kinase-Aktivitäten unter verschiede-nen physiologischen und pathophysiologischen Bedingungen. Ein weiteres Projekt, die Untersuchung der Alpha-Synuclein-Struktur innerhalb von Zellen, dient dazu, die Parkinson-Krank-heit auf molekularer Ebene zu verstehen.

S T R U C T U R A L B I O L O G Y S E C T I O N B E R E I C H S T R U K T U R B I O L O G I E

34 35

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2R E S E A R C H G R O U P S / / / S T R U C T U R A L B I O L O G Y F O R S C H U N G S G R U P P E N / / / S T R U K T U R B I O L O G I E

N M R - S U P P O R T E D S T R U C T U R A L B I O L O G Y

N M R - U N T E R S T Ü T Z T E S T R U K T U R B I O L O G I E

G R O U P L E A D E RP R O F. D R . H A R T M U T O S C H K I N AT

B I O G R A P H Y

1975 – 1976 Chemistry degree at the University of Frankfurt and 1978 – 1983 Chemistry degree at the University of Frankfurt; Diploma 1983 – 1984 Visit to the laboratory of Prof. Dr. Ray Freeman, Oxford, England 1983 – 1985 Completion of dissertation in Prof. Kessler‘s Laboratory at the University of Frankfurt 1986 Graduate thesis: „Analysis of the conformation of Cyclosporin in solution using NMR-spectroscopy: development and use of new methods“ 1986 – 1987 Postdoctoral work with Prof. Dr. Bodenhausen at the University of Lausanne, Switzerland 1987 – 1991 Position as NMR-spectros- copist at the Max-Planck-Institute for Biochemistry (Martinsried, Germany), first in the Clore/Gronenborn group, later independently in the department of Prof. Huber 1992 Habilitation in Biophysical Chemistry at the Technical University of Munich 1992 – 1998 Group leader at the EMBL, Heidelberg Since 1998 Head of the department „NMR-supported Structural Biology“ at the Leibniz-Institut für Molekulare Pharmakologie, Professor of Structural Chemistry at the Free University in Berlin

S U M M A R Y

Magic-angle-spinning (MAS) solid state NMR delivers high-resolution structural informa-tion on complex, heterogeneous samples, independent of their molecular weight and without depending on crystallization. It is an attractive method for structural investiga-tions of “difficult” systems such as proteins embedded in lipid bilayers or attached to the cytoskeleton. We aim, in the long run, to carry out structural investigations within the ‘real space’ of a cell, capitalizing on a 20 – 100 fold increase in signal-to-noise through dynamic nuclear polarization (DNP). For this purpose, we are improving and testing DNP methods on biological samples in investigations on the nascent chain emerging from the ribosome and of membrane proteins in native lipid environments. Our short-term aims include de-termining the structures of membrane-integrated or cytoskeleton-attached proteins to unravel their mechanisms of action. In particular, we are investigating the transport cycle of an ABC-transporter, proton transport in channel rhodopsin, and bacterial membrane proteins such as OmpG and YadA. Furthermore, we investigate protein systems involved in protein homeostasis, including small heat shock proteins, and initial folding events of the nascent chain on the ribosome.

Z U S A M M E N FA S S U N G

Die „Magic-Angle-Spinning” (MAS)-Festkörper-NMR-Spektroskopie liefert strukturelle Informationen zu komplexen, heterogenen Proben in hoher Auflösung, und zwar unab-hängig von ihrem Molekulargewicht und ohne vorherige Kristallisation. Es handelt sich damit um eine attraktive Methode, die Struktur von schwierig zu untersuchenden Syste-men wie beispielsweise in Lipid-Doppelschichten eingebauten oder an das Zytoskelett gebundene Proteine zu untersuchen. Unser langfristiges Ziel ist es, Strukturuntersuchun-gen innerhalb des „realen Raums” einer Zelle durchzuführen, wobei als Voraussetzung durch dynamische Kernpolarisation (dynamic nuclear polarization, DNP) eine 20 –100-fache Verbesserung des Signal-Rausch-Verhältnisses erreicht wird. Zu diesem Zweck opti-mieren und testen wir DNP-Methoden an biologischen Proben, z.B. in Untersuchungen von am Ribosom wachsenden Proteinketten und von Membranproteinen in ihrer nativen Lipidumgebung. Zu den kurzfristigen Zielen gehört die Strukturaufklärung membraninte-grierter oder zytoskelettassoziierter Proteine, um ihre Wirkungsmechanismen aufzuklären. In diesem Zusammenhang untersuchen wir den Transportzyklus eines in Lipid-Doppel-schichten integrierten ABC-Transporters, den Protonentransport in Kanalrhodopsin und bakterielle Membranproteine wie OmpG und YadA. Weiterhin untersuchen wir Protein-systeme, die an der Proteinhomöostase beteiligt sind, u.a. kleine Hitzeschockproteine, sowie die initialen Faltungsereignisse wachsender Proteinketten am Ribosom.

D E S C R I P T I O N O F P R O J E C T S

Dynamic Nuclear Polarization Methods enabling structural studies of membrane-integrated receptor systems without the necessity of protein purification or the structure determination of proteins bound to the cyto-skeleton offer attractive prospects in structural biology. Dynamic nuclear polarization (DNP) magic angle spinning NMR allows the investigation of such systems, delivering the required sensitivity. With this method, the very strong polarization of electron spins is transferred to nuclear spins, which can then be detected at much higher signal-to-noise. We have a number of ongoing stud-ies using DNP and involving crystalline preparations of soluble proteins (SH3 domain), membrane proteins (neurotoxin II bound to the nicotinic acetyl choline receptor, OmpG, mistic), amyloid fibrils (Stefin B) and even selectively labeled ribosomes. Initial results from these studies include the enhancement of sensitiv-ity through additional application of protein deuteration, studies of the effects of low temperatures on protein structures, and first assignments of residues in the nascent protein chain emanating from the ribosome. In a nutshell, the enhancements achieved by DNP make possible experiments that were previously out of reach; however the situation is complicated by a multiplica-tion of resonance lines in the spectra known as ‘heterogeneous broadening’. This observation calls for an improvement of sam-ple preparation conditions – e.g. applying shock freezing tech-niques and transfer of the sample in a frozen state into the mag-net – and the spectroscopic means of handling this situation. In one of these pilot studies we were able to solve some of these problems. We observed well-resolved solid-state NMR spectra of extensively 13C labeled neurotoxin II bound to the nicotinic acetylcholine-receptor (nAChR) in native membranes (Fig. 1, p. 36). We saw that TOTAPOL, a biradical required for DNP, localizes at the membrane and protein surfaces. The concentration of active, membrane-attached biradical decreases with time, probably due to reactive components of the membrane preparation. An optimal distribution of active biradical has powerful effects on the NMR-data. The presence of inactive TOTAPOL in membrane-proxi-mal situations, but with active biradical in the surrounding water/glycerol ‘glass’, leads to well-resolved spectra that concomitantly benefit from a considerable enhancement (e=12). The resulting spectra are, to our knowledge, the first to show resolved signals of a protein ligand bound to receptors in native membrane patches while applying DNP conditions.

Structures of membrane proteins in native lipids by solid-state NMRSeveral membrane protein systems are investigated in our lab. We prepared the ABC-transporter ArtMP from geobacillus stearother-mophilus to investigate in detail the structural changes upon ATP hydrolysis during the transport cycle in a native lipid environment. Deuterated samples of ArtMP have been prepared, and CP- and J-coupling-based HN-correlations were showing either the signals of the whole transporter or selectively the ATP binding cassette protein, respectively. This offers tremendous potential for func-tional studies by allowing for the editing of subunits on a simple spectroscopic level. As a pilot project for developing a structure determination concept suitable for medium-sized membrane pro-teins, we investigate the structure of OmpG in lipid bilayers. A number of studies concerning labeling strategies, flexibility of the loop forming the lid of the porin, and of suitable pulse sequences, are ongoing. A structure was determined of the trimeric autotrans-porter adhesin A (YadA) from Yersinia enterocolitica Adhesin A (Fig. 2, p. 37). Many members of this family are important pathogenic-ity factors that mediate adhesion to host cells and tissues in such diverse diseases as intestinal infections causing diarrhea, urinary tract infections, or airway infections. As a result of the collabora-tion between the FMP (Barth van Rossum) and the MPI for De-velopmental Biology, Tübingen (Dirk Linke, Michael Habeck), the solid-state NMR structure of the transmembrane domain of YadA was obtained. For the NMR experiments, a single, uniformly 13C and 15N-labeled microcrystalline sample from failed crystallization trials was used. Solid-state NMR provided information on flexibility and mobility of parts of the structure, which in combination with evolutionary conservation information allowed for new insights into the autotransport mechanism of YadA. In future, considerable focus will be placed on the investigation of proton transport in Channelrhodopsin, an important protein used in neurobiology for activating nerve cells.

Interfering with protein-protein interactions In a third line of projects, we search for small-molecule inhibitors of protein-protein interactions, using PDZ (PSD-95, Dlg, ZO-1) do-mains as an example. They play important roles in cellular signal-ing pathways and are structurally characterised by a hydrophobic pocket surrounded by a conserved sequence motif, G-L-G-F. This pocket binds the C-termini of target proteins, in most cases recep-tors and ion channels. Their functional diversity and characteristic, relatively small binding pocket, make them attractive targets for the design of small-molecule inhibitors which may, in the long run,

Trent Franks,

Ümit Akbey,

Sascha Lange,

Barth-Jan van Rossum

36 37

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2R E S E A R C H G R O U P S / / / S T R U C T U R A L B I O L O G Y F O R S C H U N G S G R U P P E N / / / S T R U K T U R B I O L O G I E

G R O U P M E M B E R S Dr. Ümit Akbey Dr. Linda Ball * Dr. Benjamin Bardiaux * Dr. Anne Diehl Dr. Frank Eisenmenger Dr. Trent Franks Dr. Matthias HillerAndy Nieuwkoop Dr. Marcella Orwick Dr. Barth van Rossum Dr. Monica Santos de Freitas * Shakeel Shahid

Anup Chowdhury (doctoral student) Nestor Kamdem (doctoral student) Britta Kunert (doctoral student) * Sascha Lange (doctoral student) Arne Linden (doctoral student) * Stefan Markovic (doctoral student) * Gregorio Giuseppe de Palma (doctoral student) *Joren Sebastian Retel Florian Seiter (doctoral student) Anja Voreck (doctoral student) Anne Wartenberg (doctoral student)

Janet Zapke (doctoral student) * Nils Cremer (technical assistant) * Natalja Erdmann (technical assistant) Lilo Handel (technical assistant) Martina Leidert (technical assistant) Thi-Bich Nguyen (technical assistant) * Kristina Rehbein (technical assistant)

Group members as of 31.12.2012 * Part of reporting period

Fig. 1: Simulated model

of Neurotoxin II (PDB

1NOR, light blue) bound

to nACh-receptors (gray)

in native membranes

(dark blue). The biradical,

required for DNP-NMR,

is inactive (non-luminous

orange rods) at the

surfaces of the analyte,

but still active (luminous

orange rods) in the

surrounding solvent.

This biradical distribution

leads to enhancements

of well-resolved signals.

Fig. 2: Solid-state NMR structure of the bacterial

adhesin YadA from Yersinia enterocolitica.

Shakeel Shahid et. al, Nature Methods, 2012

S E L E C T E D P U B L I C AT I O N S Bardiaux B, van Rossum BJ, Nilges M, Oschkinat H (2012) Effi-cient modeling of symmetric protein aggregates from NMR data. Angew Chem Int Ed 51: 6916-6919.

Jehle S, Vollmar BS, Bardiaux B, Dove KK, Rajagopal P, Gonen T, Oschkinat H, Klevit RE (2011) N-terminal domain of alphaB-crystallin provides a conformational switch for multimerization and structural heterogeneity. Proc Natl Acad Sci USA 108: 6409-6414.

Köhler C, Lighthouse JK, Werther T, Andersen OM, Diehl A, Schmieder P, Du J, Holdener BC, Oschkinat H (2011) The struc-ture of MESD45-184 brings light into the mechanism of LDLR fam-ily folding. Structure 19: 337-348.

E X T E R N A L F U N D I N G Deutsche Forschungsgemeinschaft, SFB 765/C4, „Multivalen-te Protein-Protein-Interaktionen zwischen WW-Domänen und Prolin-reichen Segmenten“, with Christian Freund, 01.08 – 12.11, 291.200 Euro Deutsche Forschungsgemeinschaft, SFB 740/B07-1, „Untersuchun-gen an Komplexen kleiner Hitzeschockproteine mit Substraten mittels Festkörper-NMR-Spektroskopie und dynamischer Kern-polarisation“, with B. van Rossum, 01.11 – 12.14, 497.500 Euro Deutsche Forschungsgemeinschaft, FOR 806/05, „Modulation of PDZ-domain-mediated protein-protein interactions“, OS 106/7-2, With G. Krause and J. Rademann, 01.10 – 02.13, 340.000 Euro Deutsche Forschungsgemeinschaft, FOR 806/Z2, Administration and central services, OS 106/11-2, 05.2010 – 04.2013, 145.250 Euro Deutsche Forschungsgemeinschaft, „DIP – Dynamic Nuclear Polarization: Integrating fundamentals and new applications“, OS 106/12-1, 01.2011 – 03.2014, 198.600 Euro

European Union, 7. Framework Programme, project within Bio-NMR „NMR for Structural Biology“, FP7-Infrastructures-2010-261863 01.09.2010 – 31.08.2014, 597.972 Euro European Union, 7. Framework Programme, project within EDICT “European Drug Initiative on Channels and Transporters”, EDICT/Health-F4-2007-201924, 02.2008 – 01.2012, 179.325 Euro European Union, 7. Framework Programme, project within Struc-tural Biology of Membrane Proteins (SBMPs), SBMPs/PITN-GA-2008-211800, 09.2008 – 08.2012, 475.244 Euro European Union, 7. Framework Programme, „Exploiting the Potential of Structural Biology through NMR and Associa-ted Technologies. Opportunities for the Economic Develop-ment of the Biotechnology and Pharmaceutical Industries in Tuscany, Berlin-Brandenburg and Beyond.“, EPISODE/FP7-REGI-ONS-2008-229761, 01.2009 – 31.12.2011, 107.505 Euro

Lalli D, Schanda P, Chowdhury A, Retel J, Hiller M, Higman VA, Handel L, Agarwal V, Reif B, van Rossum B, Akbey U, Oschkinat H (2011) Three-dimensional deuterium-carbon correlation experi-ments for high-resolution solid-state MAS NMR spectroscopy of large proteins. J Biomol NMR 51: 477-485.

Linden AH, Lange S, Franks WT, Akbey U, Specker E, van Ros-sum BJ, Oschkinat H (2011) Neurotoxin II bound to acetylcholine receptors in native membranes studied by dynamic nuclear polar-ization NMR. J Am Chem Soc 133: 19266-19269.

FMP authorsGroup membersC O L L A B O R AT I O N S

International Olav M. AndersenUniversity of Aarhus, Denmark Burkhard BechingerUniversity of Strasbourg/CNRS, France Teresa CarlomagnoEuropean Molecular Biology Laboratory, Heidelberg, Germany Lyndon EmsleyENS Lyon, France Robert G. GriffinMassachusetts Institute of Technology, Cambridge, USA Victoria A. HigmanUniversity of Oxford, Oxford, UK Bernadette C. HoldenerStony Brook University, Stony Brook, USA Rachel KlevitUniversity of Washington, Seattle, USA Ernest LaueUniversity of Cambridge, Cambridge, UK

Niels Chr. NielsenCenter for Insoluble Protein Structures (inSPIN), Interdisciplinary Nanoscience Center (iNANO) and Aarhus University, Denmark Michael NilgesInstitut Pasteur, Paris, France Melanie RosayBruker BioSpin, Billerica, USA Paul TordoAix-Marseille Université, France Shimon VegaThe Weizmann Institute of Science, Rehovot, Israel

National Michael HabeckMax Planck Institut für Intelligente Systeme, Tübingen

Sandro KellerTechnische Universität Kaiserslautern, Kaiserlautern Dirk LinkeMax-Planck-Institut für Entwicklungs- biologie, Tübingen Dieter OesterheltMax-Planck-Institut für Biochemie, MartinsriedJörg RademannUniversität Leipzig, LeipzigBernd ReifTechnische Universität MünchenChristiane RitterHelmholtz Centre for Infection Research, BraunschweigHans-Günther SchmalzUniversität zu Köln, Köln

allow for the treatment of several PDZ-related human disorders such as neuropathic pain, congenital diseases, psychiatric disorders, and cancer. A large number of high-resolution structures of PDZ-ligand complexes provide an excellent basis for rational design. Inhibitors with low to medium affinity for several PDZ domains were identified and members of the respective substance classes were

collected in a ‘PDZ-library’. Improvements of the AF6 PDZ domain inhibitors by modeling-chemistry cycles resulted in compounds with ten to twenty micromolar dissociation constants, disrupting the AF6-Bcr interaction in cell lysates. We intend to exploit our re-sults further by targeting three PDZ domains (AF6, DVL, Shank3), as we seek an understanding of the biology of the respective proteins.

Ümit Akbey,

Barth-Jan van Rossum

Liselotte Handel,

Anja Voreck

38 39

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2R E S E A R C H G R O U P S / / / S T R U C T U R A L B I O L O G Y F O R S C H U N G S G R U P P E N / / / S T R U K T U R B I O L O G I E

P R O T E I N E N G I N E E R I N G

G R O U P L E A D E RP R O F. D R . C H R I S T I A N F R E U N D

B I O G R A P H Y

1983 – 1989 Studied chemistry in Düsseldorf and München 1990 – 1994 Ph.D. at the Max-Planck-Institute of Biochemistry, Martinsried (Dr. Tad Holak, Dr. Wolfram Bode) 1994 – 1997 Post-Doc at the University of Zürich (Prof. Plückthun), Zürich 1997 – 2000 Post-Doc at Harvard Medical School (Prof. Wagner and Prof. Reinherz), Boston 2000 – 2011 Group Leader at the FMP 2011 – 2012 Professor of Biochemistry, Freie Universität Berlin

S U M M A R Y

Our group is interested in understanding and manipulating the molecular interactions that govern the assembly of protein complexes. Our focus is on scaffolding proteins that mediate non-covalent interactions in immune cells and other eukaryotic cell types. We use NMR spectroscopy to obtain information at atomic resolution and this can be used to rationalize individual protein-ligand pairs. Complementary methods, including phage display, peptide SPOT analysis and spectroscopy, provide valuable information on rec-ognition codes and potential in vivo interactions. Spatial information is also used for the rational and semi-rational design of alternative scaffolds with novel binding properties. We also use adaptor domains as baits in pulldown experiments. In combination with SILAC-MS and site-specific inhibition of protein interaction sites, we can assess the rela-tive importance of individual domains to the modular assembly of protein complexes. By examining adaptor domains that have undergone post-translational modifications, we aim to model and understand the dynamic regulation of specific signal transduction pathways in the cell. This has led our research to the functional investigation of antigen presentation, T cell adhesion and mRNA surveillance.

Z U S A M M E N FA S S U N G

Unsere Gruppe interessiert sich für die molekularen Wechselwirkungen, die für den Zusammenbau von Proteinkomplexen verantwortlich sind, möchte sie verstehen und manipulieren können. Dabei konzentrieren wir uns auf Gerüstproteine, die nicht-kovalen-te Wechselwirkungen in Immunzellen und anderen eukaryotischen Zelltypen vermitteln. Mit Hilfe der NMR-Spektroskopie erhalten wir genaue Strukturinformationen in atomarer Auflösung, die es erlauben, individuelle Protein-Ligand-Paare zu verstehen. Ergänzende Methoden wie Phagen-Display-Technik, Peptid-SPOT-Analyse und Spektroskopie liefern wertvolle Informationen zu Erkennungsstrukturen und möglichen in vivo-Wechselwirkun-gen. Für den rationalen und semi-rationalen Entwurf solcher Gerüstproteine mit neuen Bindungseigenschaften werden diese räumlichen Informationen ebenfalls verwendet. In Pulldown-Experimenten nutzen wir auf der Suche nach Interaktionspartnern Adapterdo-mänen als Köder („baits“). In Kombination mit SILAC-MS und spezifischer Hemmung von Protein-Wechselwirkungs-Stellen können wir die relative Bedeutung individueller Domä-nen für den modularen Zusammenbau von Proteinkomplexen beurteilen. Anhand der Untersuchung von Adapter-Domänen, die posttranslational modifiziert wurden, versu-chen wir, die dynamische Regulation spezifischer Signaltransduktionswege in der Zelle zu modellieren und zu verstehen. Dies hat unsere Forschung in Richtung funktionaler Unter-suchung von Antigen-Präsentation, T-Zell-Anheftung und mRNA-Überwachung geführt.

D E S C R I P T I O N O F P R O J E C T S

Integrin regulation in immune cellsThis project seeks to elucidate the role that posttranslational mod-ifications of the adaptor proteins adhesion and degranulation-promoting adaptor protein (ADAP) and Src kinases-associated protein of 55 kD (SKAP55) and the role they play during inside-out signaling – the signaling pathway responsible for integrin activa-tion and subsequent T-cell adhesion to the APC – in T cells. Tyro-sine and serine phosphorylation, as well as attachment of lipids like palmitate to cysteine residues, serve as a means to reversibly regulate the membrane binding properties and modular structure of components of the ADAP complex, while SKAP55 itself revers-ibly interacts with membranes via its pleckstrin homology (PH) do-main. Proteomic analysis of individual domains of ADAP allowed us to delineate the composition of the complex and unmasked functional links to the cytoskeleton and certain kinase pathways.

MHC-peptide interactionsThe central event of CD4+ T cell mediated immunity is the rec-ognition of MHCII:peptide complexes by the clonotypic T cell receptor. Despite the vast knowledge on the structure and func-tion of MHC molecules, essential mechanistic insights are lacking in regard to antigen loading and exchange. In acidic endosomal vesicles MHCII proteins (as for example the allele HLA-DR1) pre-loaded with the placeholder CLIP (class II associated invariant chain peptide) encounter antigenic peptides. HLA-DM (human leukocyte antigen DM, a non-classical MHC molecule lacking a functional peptide binding groove) catalyses exchange of CLIP for higher affinity antigens in a mechanism not yet understood. We use NMR spectroscopy to ask which structural elements pre-dispose MHCII molecules to transient HLA-DM encounter. Fur-thermore we found that unusual peptide binding modes exist and we have successfully engineered MHCII molecules with ori-entational preferences. Currently we are investigating the physi-ological role of such non-canonical antigen-presenting complexes.

Proline-rich sequence recognitionThe interaction between proline-rich sequence recognition do-mains (PRDs) and their ligands is characterized by low affinity with correspondingly high off-rates and by moderate specific-ity favoring transient binding. Dynamic binding events com-prising PRD and PRS are pivotal for the splicing of pre-mRNA. PRD-containing proteins of the spliceosome include, amongst others, the GYF-domain containing protein CD2BP2, which could be detected in the U5snRNP interacting with U5-proteins and in the U2 snRNP where it interacts with proline-rich pro-teins, and the WW-domain containing protein FBP21 which has been implicated in bridging U1 and U2 snRNPs during the tran-sition from complex E to complex A. Both proteins, CD2BP2 and FBP21, are, to a certain extent, recruited to the same mo-lecular complexes via proline-rich sequence hubs such as the core spliceosomal protein SmB/B’. In our group we investigate the functional consequences of CD2BP2/FBP21 ablation in liv-ing cells and their impact on constitutive and alternative splic-ing. In parallel we perform mechanistic studies that question the role of multivalent PRS binding motifs for spliceosome assembly.

Synaptic vesicle clusteringSignaling between neuronal cells is based on the exocytosis of neurotransmitters-filled vesicles at the active zone of the synapse. To maintain an adequate level of neurotransmission synaptic vesi-cles have to be endocytosed, recycled and exocytosed again in a process called the ‘synaptic vesicle cycle’. This cycle requires the involvement of several proteins often arranged in specific scaf-folds. One adaptor protein that is a component of scaffolds acting in multiple steps of the cycle, including endocytosis and vesicle clustering, is Intersectin 1 (ITSN1). Our aim, as part of the Sfb 958, is the investigation of the SH3 domain dependent protein-protein interactions and regulatory mechanisms that facilitate the versatile role of ITSN1 in the synaptic vesicle cycle. To achieve this, we apply different protein biochemical and structural biologi-cal methods, including sortase-mediated ligation, phage display, NMR spectroscopy and ITC.

Sebastian Günther,

Daniela Kosslick

40 41

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2

S E L E C T E D P U B L I C AT I O N S Kliche S, Worbs T, Wang X, Degen J, Patzak I, Meineke B, Togni M, Moser M, Reinhold A, Kiefer F, Freund C, Förster R, Schraven B (2012) CCR7-mediated LFA-1 functions in T cells are regulated by 2 independent ADAP/SKAP55 modules. Blood 119: 777-785. Piotukh K, Freund C (2012) A novel hSH3 domain scaffold engi-neered to bind folded domains in CD2BP2 and HIV capsid protein. Protein Eng Des Sel 25: 649-656. Schlundt A, Günther S, Sticht J, Wieczorek M, Roske Y, Heinemann U, Freund C (2012) Peptide linkage to the alpha-subunit of MHCII creates a stably inverted antigen presentation complex. J Mol Biol 423: 294-302. Klippel S, Wieczorek M, Schümann M, Krause E, Marg B, Seidel T, Meyer T, Knapp E W, Freund C (2011) Multivalent binding of formin-binding protein 21 (FBP21)-tandem-WW domains fosters protein recognition in the pre-spliceosome. J Biol Chem 286: 38478-38487. Piotukh K, Geltinger B, Heinrich N, Gerth F, Beyermann M, Freund C, Schwarzer D (2011) Directed evolution of sortase A mutants with altered substrate selectivity profiles. J Am Chem Soc 133: 17536-17539.

FMP authorsGroup members

E X T E R N A L F U N D I N G Deutsche Forschungsgemeinschaft, SFB 765, C04, “Multivalen-te Protein-Protein-Interaktionen zwischen WW-Domänen und Prolin-reichen Segmenten”, jointly with H. Oschkinat (FMP Ber-lin), 01.2012 – 12.2015, 175.200 Euro Deutsche Forschungsgemeinschaft, SFB 958, A07, “Regulation of SH3 domain-containing scaffolds in synaptic vesicle clus-tering”, jointly with V. Haucke (FMP Berlin), 07.2011 – 06.2015, 203.200 Euro Deutsche Forschungsgemeinschaft, „Mechanismus des durch HLA-DM und kleine Moleküle vermittelten MHCII:Peptid-Aus-tauschs“, FR 1325/11-1, 07.2011 – 06.2014, 122.600 Euro Leibniz-Gemeinschaft, “Development of Novel NMR Probes: Improving Cell Profiling for Early Diagnosis senates internal competition”, SAW project, jointly with L. Schröder (FMP Berlin), 01.2011 – 31.12.2013, 232.800 Euro Deutsche Forschungsgemeinschaft, FOR 806, “Analysis and inhi-bition of GYF domain mediated protein interactions”, 02.2010 – 01.2013, 310.800 Euro Bundesministerium für Bildung und Forschung (BMBF), Innova-tionspreis Medizintechnik, „Biosensor-basierte 129Xe-Magnetre-sonanztomographie in Zellen und Mausmodellen der Autoim-munität“, jointly with Dr. Lorenz Mitschang & Dr. Wolfgang Kilian, PTB, Berlin branch, 05.2010 – 12.2013, 186.700 Euro

Deutsche Forschungsgemeinschaft, “Assemblierung und Mem-bran-Rekrutierung des ADAP/SKAP55-Komplexes”, SFB 854, TP12 with B. Schraven, S. Kliche, 01.2010 – 12.2013, 177.600 Euro For joint projects, funding is shown for the whole funding period for my project part only.

R E S E A R C H G R O U P S / / / S T R U C T U R A L B I O L O G Y F O R S C H U N G S G R U P P E N / / / S T R U K T U R B I O L O G I E

G R O U P M E M B E R S Dr. Gesa Albert (doctoral student/post-doc) * Dr. Kirill Piotukh * Dr. Jana Sticht * Sebastian Günther (doctoral student) * Daniela Kosslick (doctoral student) * Roland Lehmann (doc toral student) * Andreas Schlundt (doctoral student) * Marek Wieczorek (doctoral student) * Stefan Klippel (doctoral student, shared with Schroeder group) Kathrin Motzny (technical assistant) *

Group members as of 31.12.2012 * Part of reporting period

Fig. 2: ADAP primary structure. Phosphorylation-

sites and known binding partners to each position

are highlighted.

C O L L A B O R AT I O N S International Christoph WülfingUniversity of Bristol, United Kingdom Ellis ReinherzHarvard Medical School, Harvard University, Boston, USA Coro Paisan-Ruiz Mount Sinai School of Medicine, New York, USA John GrossUniversity of California, San Francisco, USA Balaji Prakash Indian Institute of Technology Kanpur, India

National Burkhart Schraven Otto-von-Guericke University Magdeburg Udo HeinemannMax-Delbrück Center for Molecular Medicine, Berlin Dirk SchwarzerEberhard Karls Universität Tübingen Klaus-Peter KnobelochUniversitätsklinikum Freiburg Ulrich KalinkeTwincore, Hannover

Fig. 1: (A) Proline-rich recognition domains that are investigated in our la-

boratory. Shown are representative structures of an SH3 (PDB code: 1PRM),

a WW(1EG4) and a GYF domain (1L2Z) in complex with a respective ligand.

(B) CD2BP2-GYF and FBP21-WW play a role in the assembly of the early

spliceosome, thereby modulating (alternative) splicing.

Andreas Schlundt

Gesa Albert

Fabian Gerth

42 43

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2R E S E A R C H G R O U P S / / / S T R U C T U R A L B I O L O G Y F O R S C H U N G S G R U P P E N / / / S T R U K T U R B I O L O G I E

S T R U C T U R A L B I O I N F O R M AT I C S A N D P R O T E I N D E S I G N

S T R U K T U R - B A S I E R T E B I O I N F O R M AT I K U N D P R O T E I N D E S I G N

G R O U P L E A D E RD R . G E R D K R A U S E

B I O G R A P H Y

1970 – 1975 Studied chemistry at the University in Leipzig 1982 Ph.D. in biochemistry at the Martin Luther University, Halle 1981 – 1986 Researcher at the Institute of Drug Design, Berlin on behalf of pharmaceutical industry 1986 – 1991 Research Position at the Institute of Drug Design, Berlin 1991 – 1992 Visiting Scientist at the Washington University (Prof. Marshall) in St. Louis, MO, USA 1992 – 1997 Project leader at research institute of molecular pharmacology (FMP), Berlin since 1998 Group Leader of Structural Bioinformatics and Protein Design at the FMP

S U M M A R Y

The group focuses on the analysis of the relationship between sequences and struc-tures of membrane proteins using structural bioinformatics, combined with ex-perimental studies of the functions of altered sequence(s). Our aim is to reveal the structure-function relationships of proteins and potential interaction partners. One major activity is to develop bioinformatic tools for investigating such structure-func-tion relationships; another is to apply them to particular molecular biological proj-ects of protein-ligand, protein-substrate or protein-protein interactions. To verify our structure-function hypotheses we perform experimentally site-directed mutagenesis of specific residues and analyze available mutation data. Bioinformatic tool/data-base development and molecular biology applications mutually support one another. The main aims of the group are to achieve: (1) A detailed understanding of intramolecular mechanisms of membrane proteins; (2) The rational discovery of molecular mechanisms and sites for protein-protein interactions and protein-ligand or protein-substrate interac-tions; and (3) A narrowing down of locations for potential pharmacological interventions to the amino acid and atomic level.

Z U S A M M E N FA S S U N G

Die Gruppe beschäftigt sich mit der Analyse der Beziehung zwischen Sequenz und Struktur von Membranproteinen; dies erfolgt mittels struktureller Bioinformatik, in Kom-bination mit experimentellen Funktionsuntersuchungen von gezielt veränderten Prote-insequenzen. Unser Ziel ist es, Struktur-Funktionsbeziehungen von Proteinen und poten-ziellen Interaktionspartnern aufzuklären. Einerseits werden bioinformatische Werkzeuge und Datenbanken zur Untersuchung solcher Struktur-Funktionsbeziehungen entwickelt; andererseits werden diese bei spezifischen molekularbiologischen Projekten wie z.B. allo-sterische Ligandenbindung an G-Protein gekoppelte Rezeptoren, und der Wechselwir-kung von Enterotoxinen an Tight junction Proteinen eingesetzt. Zur Überprüfung unse-rer Struktur-Funktions-Hypothesen führen wir zielgerichtete Mutagenesen an spezifischen Resten experimentell durch und analysieren publizierte Mutationsdaten. Die Entwicklung von Bioinformatikwerkzeugen/Datenbanken und experimentelle Molekularbiologie unter-stützen sich dabei gegenseitig. Die Hauptziele, die wir mit unserer Gruppe erreichen wol-len, sind folgende: (1) ein detailliertes Verständnis der intramolekularen Mechanismen von Membranproteinen; (2) die rationale Aufdeckung der molekularen Mechanismen und Lokalisationen von Protein-Protein-Interaktionen und Protein-Ligand- bzw. Protein-Subs-trat-Interaktionen; und (3) eine Einengung der in Frage kommenden Interaktionspartner für potenzielle pharmakologische Interventionen auf Aminosäure- und atomarer Ebene.

D E S C R I P T I O N O F P R O J E C T S

Development of structural bioinformatic tools Of central importance to elucidate structural-functional properties of the wild-type receptor is the fact that amino acid side-chain substitutions often modify the phenotypes. On the other hand, throughout evolution each G-protein coupled receptor (GPCR) underwent a gradual change over time by mutation and natural selection. Such evolutionary processes leave their signatures in the sequence variation of extant species (orthologs). Therefore a comprehensive set of orthologous sequences of a GPCR allows us to predict the functional relevance of mutations for each position. We have generated a new web system for evaluating the molecu-lar effects of genetic variations on the basis of their evolutionary relationship at the example of the ADP receptor (P2Y12 mutant library; www.ssfa-7tmr.de/p2y12). The feasibility of this approach was verified by comparing the evolutionary conservation of avail-able 70 ortholog sequences with experimental data of a com-prehensive in vitro mutant library (site-saturation mutagenesis of every possible substitution at 66 contiguous positions covering transmembrane helices 6 and 7, =1254 mutants) (Cöster et al., 2012). To assess the utility of our approach we additionally ana-lyzed genetic variants of P2Y12 known to cause platelet defects. For this purpose we generated initial homology models for all P2Y12 orthologs using our new web accessible pipeline for GPCR homology modelling (www.ssfa-7tmr.de/ssfe) that stores the tem-plate predictions, sequence alignments, identified sequence and structure motifs and homology models for 5025 family A GPCRs (Worth et al., 2011). The impact of a particular mutation was evalu-ated by comparing the sequence variation of ortholog species with the structural space surrounding a specific natural variant. We utilized the huge amount of functional data available from both naturally occurring and designed mutations (> 1100) of the glycoprotein hormone receptors (GPHRs). The recent complete renewal and extension of a previous database version (www.ssfa-GPHR.de) included the addition of several new features, such as an interactive 3D search and a new section for double and triple mutations in addition to single mutations (Kreuchwig et al., 2011).

Sequence-Structure-function-relationships at GPCRs Such relationships were deciphered for i) signalling effects of li-gands at GPCRs (Wichard et al., 2011; cooperation with R Kühne), ii) allosteric effects of small molecules at LHR (Heitman et al., 2012), iii) structural determinants of the TSH-receptor for signal-ling (Kleinau et al., 2011a, Kleinau et al., 2011b; cooperation with R. Schülein (Teichmann et al., 2012)) and iv) molecular details of activation and inactivation patterns at the transmembrane domain by mutagenesis sampling of residues that cover a potential al-losteric ligand binding pocket of the thyrotropin receptor, TSHR (Haas et al., 2011). Clostridium perfringens enterotoxin (CPE) variants targeting claudinsThe latter successful modelling driven mutations motivated us to adopt this strategy for the interacting interface between CPE and claudins, the major constituents in tight junctions (TJ). CPE has prompted interest in TJ research since it is as of yet the only known extracellular high affinity (KD ~10 nM) native ligand of claudins. CPE binds to the second extracellular loop 2 (ECL2) of distinct claudins (claudin-3, -4) via its C-terminal domain (cCPE), which on its own is non-cytotoxic, but increases paracellular per-meability. Our hypothesis is that the formation of paracellular barriers is hindered by claudin sequestering, probably by the bulkiness of bound cCPE. Since there is a lack of high affinity modulators for TJ to distinguish between the different claudin-subtypes, we utilized the available crystal structures for CPE and generated cCPE / claudin-ECL2 interaction models (Fig.1, p. 44) to understand the molecular mechanisms to modify cCPE as clau-din modulator targeting individual claudin subtypes in TJ barriers and other cellular contexts. We showed by model-guided muta-tions that cCPE exhibits different binding modes toward Cld3 and Cld4, which we could utilize to design cCPE constructs binding preferentially either to Cld3 or Cld4 (Fig.2, p. 44) (Veshnyakova et al., 2012). Therewith we proved the concept that modifications of cCPE allow a targeted interaction with distinct claudin subtypes for potential pharmacological interventions (cooperation with J Piontek).

Gerd Krause,

Jonas Protze,

Franziska Kreuchwig,

Annika Kreuchwig

44 45

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2R E S E A R C H G R O U P S / / / S T R U C T U R A L B I O L O G Y F O R S C H U N G S G R U P P E N / / / S T R U K T U R B I O L O G I E

G R O U P M E M B E R S Dr. Anita Kinne (doctoral student/postdoc) * Dr. Gunnar Kleinau * Dr. Anna Piontek (née Veshnyakova) * Dr. Catherine Sargent (née Worth) * Miriam Eichner (doctoral student, co-supervisor J. Piontek) * Paul Grzesik (doctoral student) * Ann-Karin Haas (doctoral student) * Katrin M. Hinz (doctoral student) * Inna Hoyer (doctoral student) * Annika Kreuchwig (doctoral student) * Jonas Protze (doctoral student) Christian Schillinger (doctoral student) *

Group members as of 31.12.2012 * Part of reporting period

Fig. 1: Details of the interaction model for the interface between the

extracellular loop2 of Claudin-3 (backbone orange, residues white) and

clostridium perfringens enterotoxin (clipped surface of binding pocket,

green, polar/charged; yellow, hydrophobic). Complementary mutations

confirmed the appropriate orientation, side chain Leu150 (orange mesh) of

Cld3-ECL2 binds into a deeper pit of the binding pocket of CPE encircled

by Y306, Y310 and Y312. Side chain Pro152 of Cld3-ECL2 point to the

smaller pit covered by L254, L315, L323.

Inna Hoyer

Jonas Protze

Fig. 2: Differing cCPE-binding to full-length Claudin-3 and Claudin-4 by

distinct mutations in cCPE. (A) Interaction models of ECL2 for Cld3 (or-

ange) and Cld4 (green) bound to binding cavity at the surface of the cCPE

structure (grey). The ECL2 helix-turn-helix models demonstrate differing

binding modes (C-terminal helix, right side) between Cld3 and Cld4 that

are consistent with the mutation data, since Cld3 is tilted towards cCPE

residues showing stronger mutational effects on Cld3 binding (cyan),

whereas Cld4 is tilted to residues exhibiting stronger effects on Cld4 bind-

ing (red). (B) Quantification cCPE-binding to living HEK cells expressing

Cld3wt or Cld4wt. Designed multiple CPE mutations strongly affect either

Cld4 (red; LDR: L223A/ D225A/R227A) or Cld3 (cyan; LSID: L254A/S256A/

I258A/D284A) that prefer binding either to Cld3 or Cld4 respectively.

S E L E C T E D P U B L I C AT I O N S Cöster M, Wittkopf D, Kreuchwig A, Kleinau G, Thor D, Krause G, Schöneberg T (2012) Using ortholog sequence data to predict the functional relevance of mutations in G-protein-coupled receptors. FASEB J 26: 3273-3281 Krause G, Kreuchwig A, Kleinau G (2012) Extended and structur-ally supported insights into extracellular hormone binding, signal transduction and organization of the thyrotropin receptor. Plos One 7: e52920 Veshnyakova A, Piontek J, Protze J, Waziri N, Heise I, Krause G (2012) Mechanism of Clostridium perfringens enterotoxin interac-tion with claudin-3/-4 protein suggests structural modifications of the toxin to target specific claudins. J Biol Chem 287: 1698-1708. Haas AK, Kleinau G, Hoyer I, Neumann S, Furkert J, Rutz C, Schülein R, Gershengorn MC, Krause G (2011) Mutations that si-lence constitutive signaling activity in the allosteric ligand-binding site of the thyrotropin receptor. Cell Mol Life Sci 68: 159-167 Kreuchwig A, Kleinau G, Kreuchwig F, Worth CL, Krause G (2011) Research resource: Update and extension of a glycoprotein hor-mone receptors web application. Mol Endocrinol 25: 707-712. FMP authorsGroup members

E X T E R N A L F U N D I N G Deutsche Forschungsgemeinschaft, SPP 1629, “Role of L-type amino acid transporter Lat2 in transport of thyroid hormones”, KR 1273/5-1, 11.2012 – 10.2015, 139.800 Euro Deutsche Forschungsgemeinschaft, SPP 1629, “Role of L-type amino acid transporter Lat2 in transport of thyroid hormones”, KI 1751/1-1, 08. 2012 – 07.2015, 227.950 Euro Deutsche Forschungsgemeinschaft, „Modulatoren für den Thy-rotropin-Rezeptor: Molekulare Mechanismen allosterischer Bindung und Wirkungsweise kleiner Moleküle“, KR 1273/4-1, 01.2010 – 09.2013, 224.250 Euro Deutsche Forschungsgemeinschaft, FOR 721, „Molekulare und strukturelle Muster parazellulärer Poren durch subtypabhän-gige Claudin-Claudin-Wechselwirkungen in tight junction“, KR 1273/3-2, with J. Piontek, 01.2010 – 09.2013, 315.840 Euro Deutsche Forschungsgemeinschaft, FOR 806 TP 5, „Modulation of PDZ-domain-mediated protein-protein interactions“, with H. Oschkinat and J. Rademann, 01.2010 – 02.2013, 342.000 Euro

C O L L A B O R AT I O N S International M. Gershengorn and S. NeumannNIH Bethesda, MD, USA A. Ijzerman and L. HeitmanUni Leiden , The Netherlands

National H. BiebermannCharité – Universitätsmedizin Berlin M. FrommCharité – Universitätsmedizin Berlin J. GromollUnivertsity Münster J. KöhrleCharité – Universitätsmedizin Berlin T. SchönebergUniversität Leipzig U. SchweizerCharité – Universitätsmedizin Berlin

46 47

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2R E S E A R C H G R O U P S / / / S T R U C T U R A L B I O L O G Y F O R S C H U N G S G R U P P E N / / / S T R U K T U R B I O L O G I E

C O M P U TAT I O N A L C H E M I S T R Y / D R U G D E S I G N

W I R K S T O F F - D E S I G N

G R O U P L E A D E RD R . R O N A L D K Ü H N E

B I O G R A P H Y

1969 – 1973 Study of Biochemistry at the Martin-Luther-Universität Halle Witttenberg 1973 Diploma in Biochemistry 1973 – 1976 Research associate at the Zentralinstitut für Molekularbiologie und Medizin der Akademie der Wissenschaften 1976 – 1992 Research associate at the Institut für Wirkstofforschung der Akademie der Wissenschaften 1980 Doctorate degree since 1993 Group leader at the Leibniz- Institut für Molekulare Pharmakologie

S U M M A R Y

The rational design of compounds which bind to biologically important target proteins integrates different scientific disciplines such as computational chemistry, bioinformatics, and molecular modeling and needs to be closely linked to an experimental examination of predicted chemical entities. The most effective way to organize this activity combines both experimental and theoretical work in one research unit. Within our group the full repertoire of in-silico methods for homology modeling, virtual screening, and ligand opti-mization is enhanced through experimental binding studies in combination with different biochemical and cell-based methods.

Z U S A M M E N FA S S U N G

Das rationale Design von Substanzen, die an biologisch wichtige Zielproteine binden, ver-eint unterschiedliche wissenschaftliche Disziplinen wie computergestützte Chemie, Bio-informatik und Molekülmodellierung und sollte eng mit einer experimentellen Untersu-chung der vorhergesagten chemischen Wirkstoffe verknüpft sein. Am effizientesten lässt sich diese Verknüpfung von Theorie und Praxis realisieren, wenn diese Arbeiten in ein und derselben Forschungseinheit erfolgen. Innerhalb unserer Gruppe wird das gesam-te Spektrum von in-silico-Methoden für Homologie-Modellierung, virtuelles Screening und Ligand-Optimierung durch experimentelle Bindungsstudien in Kombination mit ver-schiedenen biochemischen und zellbasierten Methoden erweitert.

D E S C R I P T I O N O F P R O J E C T S

Interfering with poly-proline-mediated protein-protein interactionsProtein-protein interactions are often mediated by specialised domains that represent difficult targets for ligand design. A par-ticularly prominent group of such domains interact with short proline-rich motifs. These motifs often adopt poly-proline helix II (PPII) conformation in which two proline residues (PxxP or xPPx) cannot be replaced by any biogenic amino acid. Competitive small organic compounds are, so far, not known. Therefore, we rationally designed new chemotypes to be combined appropri-ately for mimicking pairs of adjacent prolines in PPII conforma-tion. These new chemotypes were synthesized by our collaborator (H.-G. Schmalz) and were used in new peptide chimeras showing nano-molar binding to ena/VASP EVH1 domains. Subsequent investigations lead to the first low-molecular weight inhibitor of EVH1. X-ray crystallography of this compound in complex with the enah EVH1 demonstrated an optimal positioning of the inhibitor (Fig. 1, p. 48). We showed that these new compounds were able to displace the native interaction partners out of ena/Vasp family EVH1 domains. MHC class II loading enhancersMajor histocompatibility complex class II (MHCII) molecules are peptide receptors, which display protein fragments at the cell surface. MHCII molecules are involved in a number of auto-im-mune diseases, and our aim is to understand the mechanism of the antigen loading which is mainly influenced by stabilizing the receptive MHCII conformation through HLA-DM (DM). Molecu-lar dynamics simulations of the MHCII HLA-DR1 (DR) and of the catalyst DM, using an atomic and a coarse-grained force field, were performed. Our simulations predicted a DR:DM complex in which the interfaces were mostly located on the convex face of DR, suggesting that this interaction site is important for stabilization of the complex. A similar interface in the recently published crystal structure of a HLA-DO:DM complex confirms our results. Based on our models, we are able to identify specific interactions at the interface of the complex and to predict mutations that stabilize the complex. The predictions will be tested in NMR experiments by our collaborators at the Ch. Freund lab (FU Berlin).

Chemical Biology Module Cheminformatics and Database Design (Bernd Rupp)The existing FMP database of commercially available compounds is used frequently. The increasing number of compounds required a reorganization of our storage and searching strategy. The new Registration Database captures and normalizes the data of ven-dor catalogues. Based on this database we developed the non-redundant Unique Structure Database which represents a data warehouse combining vendor data, structural descriptors and in-house classification tools including our previously developed ADMET- and reactivity filters, as well as our in-house fragment-based fingerprints used for library design tasks. Furthermore, a first version Web service is in preparation, which allows one not only to search for compounds and fragments but also to combine such a search with the FMP tools to classify compounds for their usability in biological assays. Chemical Biology Module Library Design (Michael Lisurek)Computer-chemical expertise is needed in all phases of the screening process. Success in small molecule screening relies heavily on the library composition to be screened. The challenge consists in the enrichment of a screening library with bioactive compounds. Within this task we developed in-house software for the design of both all-purpose screening libraries as well as focussed libraries around hits using a fragment-based concept. The latter is based on the identification of the most common sub-structures derived by the analysis of the MDL Drug Data Report (MDDR) and the World Drug Index (WDI). This software solution is now used to address all important aspects of library design. Within the EU-OPENSCREEN project it was decided that our library design tool box be used as a core module to develop the EU-OPENSCREEN exclusion rules for library design, pre-filtering of the commercial available vendor stock catalogues, and design of a commercially available compound library. Furthermore it is the responsibility within the module to support the chemical biol-ogy projects with our expertise in modeling, docking and virtual screening.

Matthias Müller,

Matthias Barone

48 49

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2R E S E A R C H G R O U P S / / / S T R U C T U R A L B I O L O G Y F O R S C H U N G S G R U P P E N / / / S T R U K T U R B I O L O G I E

G R O U P M E M B E R S Dr. Michael Lisurek (Head Module Library Design) Dr. Daniela Müller * Dr. Robert Opitz (doctoral student until 08/2011) * Dr. Kiril Piotukh * Dr. Bernd Rupp (Head Module Database) Raed Al-Yamori (Technical Infomatics) * Matthias Barone (doctoral student) * Matthias Müller (doctoral student) *

Group members as of 31.12.2012 * Part of reporting period

Fig. 1: Comparison of FPPPP

binding to enaH EVH1 (1evh,

colored in white) with enaH

EVH1 in complex with the low-

molecular weight inhibitor. The

new chemotypes replacing the

poly-proline motif are colored in

green and magenta, respec-

tively.

S E L E C T E D P U B L I C AT I O N S Bandholtz S, Wichard J, Kühne R, Grötzinger C (2012) Molecular evolution of a peptide GPCR ligand driven by artificial neural net-works. PloS One 7: e36948. Horatscheck A, Wagner S, Ortwein J, Kim BG, Lisurek M, Beligny S, Schütz A, Rademann J (2012) Benzoylphosphonate-based pho-toactive phosphopeptide mimetics for modulation of protein tyro-sine phosphatases and highly specific labeling of SH2 domains. Angew Chem Int Ed 51: 9441-9447. Jacso T, Schneider E, Rupp B, Reif B (2012) Substrate transport activation is mediated through second periplasmic loop of trans-membrane protein MalF in maltose transport complex of Esche-richia coli. The Journal of biological chemistry 287: 17040-17049. Rupp B, Günther S, Makhmoor T, Schlundt A, Dickhaut K, Gupta S, Choudhary I, Wiesmüller KH, Jung G, Freund C, Falk K, Rötzschke O, Kühne R (2011) Characterization of structural features control-ling the receptiveness of empty class II MHC molecules. PloS One 6: e18662. Wichard JD, Ter Laak A, Krause G, Heinrich N, Kühne R, Klein-au G (2011) Chemogenomic analysis of G-protein coupled recep-tors and their ligands deciphers locks and keys governing diverse aspects of signalling. PloS one 6: e16811.

FMP authorsGroup members

E X T E R N A L F U N D I N G

Deutsche Forschungsgemeinschaft, FOR 806 TP 03, “Design and Synthesis of low-molecular weight proline-rich motif (PRM) mimetics recognized by PRM binding domains”, KU 845/2/2, 03.2010 – 03.2013, 123.000 Euro Deutsche Forschungsgemeinschaft, Mechanismus des durch HLA-DM und kleine Molküle vermittelten MHCII:Peptid-Austauschs, KU 845/3-1, 03.2012 – 03.2015, 111.140 Euro

C O L L A B O R AT I O N S International Marcel HibertUniversité de Strasbourg, France Jordi QuintanaBarcelona Science Park, Spain

National Hans-Günther SchmalzUniversität zu Köln Christian FreundFreie Universität Berlin Markus WahlFreie Universität Berlin Ulrike SteinMax-Delbrück Center for Molecular Medicine, Berlin Bayer AG, Leverkusen

Martyna Pawletta,

Lara Kuhnke

Matthias Müller,

Matthias Barone

Michael Lisurek

Daniela Müller,

Bernd Rupp

50 51

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2R E S E A R C H G R O U P S / / / S T R U C T U R A L B I O L O G Y F O R S C H U N G S G R U P P E N / / / S T R U K T U R B I O L O G I E

S O L U T I O N N M R

L Ö S U N G S - N M R

G R O U P L E A D E RD R . P E T E R S C H M I E D E R

B I O G R A P H Y

1982 – 1984 Study of Chemistry at the University of Frankfurt and 1985 – 1987 Study of Chemistry at the University of Frankfurt 1988 Diploma thesis in Prof. Kessler’s group 1989 – 1992 Ph.D. at the TU Munich (Prof. Kessler) 1992 – 1995 Post-Doc at Harvard Medical School (Prof. Wagner) since 1995 Group leader “Solution NMR spectroscopy” at the FMP

S U M M A R Y

The group focuses on the application of solution-state NMR-spectroscopic techniques to the investigation of the structure and dynamics of biomolecules at atomic resolution. The full repertoire of multidimensional NMR techniques is used in combination with appropri-ate labeling schemes and other biophysical techniques including analytical ultracentrifu-gation (AUC), isothermal titration calorimetry (ITC) and small-angle x-ray scattering (SAXS). In recent years the group has been active in the investigation of photoactive peptides and proteins; the most recent project has been the elucidation of the structural basis of the activation mechanism of the blue-light receptor YtvA. Currently our interests have shifted towards making quantitative determinations of the role of dynamics in biomol-ecules. Here we are exploring the role of mobility in the presentation of peptides by MHC class-one molecules and their recognition by T-cell receptors, exploiting the ability of NMR spectroscopy to provide such information at atomic resolution.

Z U S A M M E N FA S S U N G

Der Fokus unserer Gruppe liegt auf der Anwendung Lösungs-NMR-spektroskopischer Techniken zur Untersuchung der Struktur und Dynamik von Biomolekülen in atomarer Auf-lösung. In Kombination mit geeigneten Markierungsmethoden und anderen biophysi-kalischen Techniken einschließlich analytischer Ultrazentrifugation (AUC), isothermischer Titrationskalorimetrie (ITC) und Kleinwinkel-Röntgenbeugung (SAXS) wird das gesamte Spektrum an mehrdimensionalen NMR-Techniken eingesetzt. In den letzten Jahren hat sich die Gruppe mit der Untersuchung photoaktiver Peptide und Proteine beschäftigt; jüngstes Projekt war die Aufklärung der strukturellen Grundlagen des Aktivierungsmecha-nimus des blaulicht-sensitiven Photorezeptors YtvA. Derzeit versuchen wir die die Rolle der Dynamik von Biomolekülen quantitativ zu erfassen. In diesem Zusammenhang erfor-schen wir die Rolle der Mobilität bei der Präsentation von Peptiden durch MHC Klasse I-Moleküle und ihrer Erkennung durch T-Zell-Rezeptoren und nutzen dazu die Fähigkeit der NMR-Spektroskopie, solche Informationen in atomarer Auflösung zu liefern.

D E S C R I P T I O N O F P R O J E C T S

Structure determination and domain interaction of the photochromic protein YtvA from Bacilus subtilisFor many organisms on earth light is not only an essential source of energy but also an external stimulus that regulates develop-mental processes. It can, however, also be potentially dangerous at shorter wavelengths. Consequently, organisms have developed mechanisms to evaluate light intensity, direction, duration and color. This is accomplished by several types of photoreceptors that usually consist of a protein component and an organic chro-mophore cofactor, often covalently attached.

We study the blue light absorbing photoreceptor YtvA from Bacillus subtilis. It has been shown that YtvA is part of the stress response in Bacillus subtilis but the detailed mechanism is not yet understood. It is located in the stressosome, a 25 S macromolecular complex responsible for the activation of the transcription regulator σB via a phosphorylation cascade and a partner switching mechanism. This photoreceptor is a two-domain protein, consisting of a LOV (light, oxygen, voltage) domain (harboring a flavin chromophore) and a STAS (Sulphate Transporters AntiSigma-factor antagonist) domain. We have used biophysical methods to obtain information on the three-dimensional structure and the changes occurring after illu-mination with blue light. Using analytical ultracentrifugation (AUC) and small-angle x-ray scattering (SAXS) we have shown that YtvA is a dimer and maintains its overall shape in the dark and after ac-tivation by blue light. We also demonstrated, based on isothermal titration calorimetry (ITC) and NMR that, contrary to data already in the literature, the function of YtvA is not to bind GTP. Most importantly the structure of YtvA was determined based on NMR spectroscopic data (Figure 1, p. 52). While a well-defined structure could be obtained in the dark, the protein becomes slightly dis-ordered after illumination with light, preventing a structure deter-mination. We are currently using ESR-techniques to obtain further information on structural changes upon illumination by blue light.

NMR spectroscopic investigation of micropolymorphism-depen-dent dynamics of human major histocompatibility antigensMHC class I molecules accommodate small peptide fragments of 8 to 12 amino acids within a binding groove and present them to T cell receptors on cytotoxic T cells. T cells constitute a necessary component of normal adaptive immune responses, but can also be involved in autoimmunity. A wealth of structural information on MHC complexes has become available, but the static picture resulting from crystallographic studies has not been able to fully explain several features of the interaction between these mol-ecules and T-cell receptors, in particular the occurrence of auto-immune reactions.

We employ heteronuclear NMR spectroscopy to investigate the dynamics of MHC complexes of HLA-B27 subtypes that differ only by a single amino acid, but are differentially associated with an au-toimmune disease. We will correlate the dynamic and structural at-tributes with distinct functional properties of these peptide-load-ed molecules. Four different MHC complexes will be investigated.

As initial steps of the project the labeling of all components of the MHC complex has to be established, and this is particularly demanding for the peptide component since large amounts of peptide will be necessary during the complex formation step. A first MHC complex with only the peptide labeled with 15N has al-ready been produced.

A second step is the assignment of all resonances in both protein components and the peptide. Given the size of the complex this requires protein deuteration, and assignment of the relatively large heavy chain is by no means trivial. Currently an assignment of b2-microglobulin in all four complexes and an assignment of the heavy chain in three of the four complexes have been achieved. Last but not least, a careful analysis of the relaxation data will be required in the present case where subtle differences between subtypes will most likely have a significant impact. Using a small protein the relaxation experiments have been optimized and soft-ware has been set up for an analysis of 15N-T1, T2 and hetero-NOE data. An example for the analysis of the dynamics of b2-micro-globulin free, and in HLA-B2709, is shown in Figure 2 (p. 52).

Matthias Dorn,

Marco Röben,

Marcel Jurk

52 53

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2R E S E A R C H G R O U P S / / / S T R U C T U R A L B I O L O G Y F O R S C H U N G S G R U P P E N / / / S T R U K T U R B I O L O G I E

G R O U P M E M B E R S Martin Ballaschk (doctoral student) Matthias Dorn (doctoral student) * Tolga Helmbrecht (doctoral student) * Marcel Jurk (doctoral student) Marco Röben (doctoral student) * Sabine Seedorff (doctoral student) * Monika Beerbaum (technical assistant) Brigitte Schlegel (technical assistant) Group members as of 31.12.2012 * Part of reporting period reported

Fig. 1: Structure of the photoreceptor YtvA

in its dark state, consisting of a LOV domain

(blue) and a STAS domain (red) connected by

the Ja-helix (green). The structure has been

determined using solution state NMR spec-

troscopy and forms the basis of a proposed

mechanism for the activation of the receptor

by blue light.

Fig. 2: Comparative analysis of the relaxation

parameters of free b2-microglobulin and b2-

microglobulin within the HLA-B2709/pVIPR MHC

complex. Distinct differences in the mobility can

be observed, and the protein becomes more

rigid upon complex formation while maintaining,

however, some flexibility at the interface to the

heavy chain.

S E L E C T E D P U B L I C AT I O N S Dorn M, Jurk M, Schmieder P (2012) Blue news update: BODIPY-GTP binds to the blue-light receptor YtvA while GTP does not. PloS One 7: e29201. Hoppmann C, Schmieder P, Domaing P, Vogelreiter G, Eich-horst J, Wiesner B, Morano I, Ruck-Braun K, Beyermann M (2011) Photocontrol of contracting muscle fibers. Angew Chem 123, 7841-7845, Angew Chem Int Ed Engl 50: 7699-7702. Jurk M, Dorn M, Schmieder P (2011) Blue flickers of hope: second-ary structure, dynamics, and putative dimerization interface of the blue-light receptor YtvA from Bacillus subtilis. Biochemistry 50: 8163-8171. Röben M, Schmieder P (2011) Assignment of phycocyanobilin in HMPT using triple resonance experiments. Magn Reson Chem 49: 543-548. Schmiederer T, Rausch S, Valdebenito M, Mantri Y, Mosker E, Bar-amov T, Stelmaszyk K, Schmieder P, Butz D, Müller SI, Schneider K, Baik MH, Hantke K, Süssmuth RD (2011) The E. coli siderophores enterobactin and salmochelin form six-coordinate silicon com-plexes at physiological pH. Angew Chem 123: 4317-4321, Angew Chem Int Ed Eng. 50: 4230-4233.

FMP authorsGroup members

E X T E R N A L F U N D I N G Deutsche Forschungsgemeinschaft, „Structure determination and domain interaction of the photochromic protein YtvA from Bacillus subtilis“, SCHM 880/8-1, 01.2009 – 12.2011, 203.400 Euro Deutsche Forschungsgemeinschaft, „NMR spectroscopic inves-tigation of micropolymorphism-dependent dynamics of human major histocompatibility antigens“, SCHM 880/9-1, 01.2011 – 12.2013, 142.000 Euro

C O L L A B O R AT I O N S National Ute CurthMedizinische Hochschule Hannover Wolfgang GärtnerMax-Planck-Institut für Chemische Energiekonversion, Mülheim an der Ruhr Enno KlussmannMax-Delbrück-Center for Molecular Medicine, Berlin Timo NiedermeyerCyano Biotec GmbH, Berlin

Martin SteupUniversität Potsdam Roderich SüssmuthTechnische Universität Berlin Barbara Uchanska-ZieglerCharité – Universitätsmedizin Berlin Andreas ZieglerCharité – Universitätsmedizin Berlin

Monika Beerbaum,

Brigitte Schlegel

Philipp Schramm,

Martin Ballaschk

54 55

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2R E S E A R C H G R O U P S / / / S T R U C T U R A L B I O L O G Y F O R S C H U N G S G R U P P E N / / / S T R U K T U R B I O L O G I E

M O L E C U L A R I M A G I N G

M O L E K U L A R E B I L D G E B U N G

G R O U P L E A D E RD R . L E I F S C H R Ö D E R

B I O G R A P H Y

1995 – 1997 Studies of Physics and Chemistry, Georg-August Universität Göttingen 1997 – 2001 Studies of Physics and Astronomy, Ruprecht-Karls Universität Heidelberg, Diploma in Physics 2001 – 2003 PhD student, Deutsches Krebsforschungszentrum and Ruprecht-Karls Universität Heidelberg, Dr. rer. nat. 2003 – 2005 Research Assistant, Deutsches Krebsforschungszentrum, Heidelberg 2005 – 2007 Emmy Noether Fellow of the DFG, University of California, Berkeley 2007 – 2009 Research Fellow, Lawrence Berkeley National Laboratory 2009 Emmy Noether Fellow of the DFG, Group Leader at FMP since 2009 ERC Starting Grantee at FMP

S U M M A R Y

Pharmacological research concerns itself with drug action and would substantially benefit from methods of imaging that directly visualize interactions between a living organism and substances that are administered. The ERC Project BiosensorImaging aims to estab-lish a novel approach to magnetic resonance imaging (MRI) to improve drug develop-ment and the monitoring of therapies. Most of today’s MRI protocols rely on detecting abundant molecules such as water and come with serious limitations on our ability to study the spatial distribution of biomarkers at low concentrations. As an alternative, xenon biosensors have an outstanding potential to improve MRI because hyperpolarized xenon for functionalized contrast agents has a 10,000-fold sensitivity compared to water. Our research focuses on in vitro and in vivo diagnostics for the localization of, for example, tumor cells and their response to drug delivery. Our interdisciplinary approach integrates aspects of atomic physics (the optimization of the hyperpolarization and NMR readout process), chemistry (the design and synthesis of targeted biosensors) and cell biology (affinity and toxicity studies). Xenon biosensors will enable a detection of molecular mark-ers at a high sensitivity without any background signal. This approach has the potential to close the sensitivity gap between modalities of nuclear medicine such as PET/SPECT and MRI without using ionizing radiation or making compromises in penetration depth that occur in optical imaging.

Z U S A M M E N FA S S U N G

Die pharmakologische Forschung beschäftigt sich mit der Wirkung von Arzneimitteln und würde von Bildgebungsverfahren, die die Wechselwirkungen zwischen einem lebenden Organismus und den verabreichten Substanzen direkt sichtbar machen, enorm profitieren. Mit dem ERC-Projekt BiosensorImaging soll ein neuer Ansatz der Magnetresonanztomo-graphie (MRT) etabliert werden, um die Wirkstoffentwicklung und Therapiekontrolle zu verbessern. Die meisten derzeitigen Verfahren basieren auf der Detektion von hochkon-zentrierten Wassermolekülen, die allerdings unsere Fähigkeit, die räumliche Verteilung von Biomarkern bei geringen Konzentrationen zu untersuchen, ernsthaft einschränken. Als Alternative hierzu verfügen Xenon-Biosensoren über ein hervorragendes Potenzial, die MRT zu verbessern, da hyperpolarisiertes Xenon und die Spezifität von funktionali-sierten Kontrastmitteln eine 10.000-mal höhere Empfindlichkeit als Wasser aufweisen. Bei unserer Forschung konzentrieren wir uns auf in vitro- und in vivo-Diagnoseverfahren, um beispielsweise Tumorzellen zu lokalisieren und ihre Reaktion auf die Wirkstoffverab-reichung zu ermitteln. In unserem interdisziplinären Ansatz vereinen wir Aspekte der ato-maren Physik (Optimierung der Hyperpolarisation und NMR-Auslese) mit der Chemie (Design und Synthese spezifischer Biosensoren) und Zellbiologie (Affinitäts- und Toxizi-tätsstudien). Xenon-Biosensoren werden eine Detektion von molekularen Markern mit hoher Sensitivität und ohne Hintergrundsignal ermöglichen. Somit haben sie das Poten-zial, die Sensitivitätslücke zwischen den Modalitäten der Nuklearmedizin wie PET/SPECT und der MRT ohne Verwendung ionisierender Strahlung oder das bei optischen Bildge-bungsverfahren oft vorkommende Eingehen von Kompromissen bezüglich der Eindring-tiefe zu schließen.

D E S C R I P T I O N O F P R O J E C T S

The LEIPNIX PolarizerThe polarization process in the newly designed, mobile LEIPNIX setup (Laser Enabled Increase of Polarization for Nuclei of Im-prisoned Xenon) is based on spin exchange optical pumping of rubidium vapor interacting with xenon and is performed with a line-narrowed laser diode at high power levels (~150 W cw op-eration). This ensures a photon density that is much higher than in conventional systems. At the same time, side effects from unwanted laser heating, e.g. the so-called ‘rubidium runaway ef-fect’, are avoided by a newly developed self-adjustable cooling mechanism at the laser entrance window. Together with optical elements that ensure improved circular polarization of the light compared to conventional setups, this allows us to work with only one compact beam path. The device yields a ~25,000-fold increase in spin polarization under continuous gas flow. In-house developed electronics allow for triggering of the gas delivery by the NMR spectrometer and ensure optimized conditions for biosensor applications with unprecedented accuracy: LEIPNIX achieves continuous production of solutions and cell suspensions saturated with hyperpolarized xenon to be detected in molecular cages with signal fluctuations < 0.4%.

Snapshot CEST ImagingInstallation of the multinuclear microimaging system was followed by NMR pulse sequence development for CEST applications (chemical exchange saturation transfer). This included pseudo-2D NMR spectroscopy for acquiring CEST spectra and characteriza-tion of 129Xe NMR encoding techniques (variable flip angle multi shot gradient echo sequences versus single shot echo planar im-aging) for Hyper-CEST imaging. In combination with the excellent performance of the LEIPNIX system, this allowed the first imple-mentation of single-shot biosensor images, pushing the sensitiv-ity limit significantly: compared to the introduction of Hyper-CEST in 2007, spatial image resolution could be improved by a factor

~4.8, while at the same time working with a 20-fold reduced sensor concentration and a 635% increase in acquisition speed. Hence, the overall 610-fold gain in sensitivity now allows detection of functionalized sensors at ~30nM concentration – a level that would usually require ~1100 years of acquisition time without the Hyper-CEST approach. These improvements also yielded the first time-resolved studies showing a dynamic contrast agent uptake with functionalized xenon and were honored with a Gorter Award by the International Society for Magnetic Resonance in Medicine.

Leif Schröder,

Chris Witte

Fig. 1: The NMR signal of hyperpolarized xenon-129 trapped in crypto-

phane cages is sensitive to its molecular environment. Smash-CEST is

a novel encoding method with optimal use of reversible xenon binding

that enables sub-second imaging for time-resolved studies (left) of sensor

uptake while maintaining high spectral selectivity (right) (animated figures

available at www.fmp-berlin.de/schroeder/smashCEST).

56 57

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2R E S E A R C H G R O U P S / / / S T R U C T U R A L B I O L O G Y F O R S C H U N G S G R U P P E N / / / S T R U K T U R B I O L O G I E

G R O U P M E M B E R S Dr. Jabadurai Jayapaul Dr. Christopher Witte Jörg Döpfert (doctoral student) Stefan Klippel (doctoral student) Martin Kunth (doctoral student) Federica Rossella (doctoral student) Matthias Schnurr (doctoral student)

Group members as of 31.12.2012

Fig. 2: Novel bimodal contrast agents allow for

combined fluorescence/NMR studies (left) to in-

vestigate interactions with biomembrane models

like giant unilaminar vesicles (right) and develop

MRI sensors for membrane fluidity, a parameter

that is also linked to cell malignancy.

S E L E C T E D P U B L I C AT I O N S Kunth M, Döpfert J, Witte C, Rossella F, Schröder L (2012) Opti-mized use of reversible binding for fast and selective NMR local-ization of caged xenon. Angew Chem Int Ed 51: 8217-8220. (hot paper, inside back cover article) Sloniec J, Schnurr M, Witte C, Resch-Genger U, Schröder L, Hen-nig A (2012) Biomembrane interactions of functionalized cryp-tophane-a: combined fluorescence and 129Xe NMR studies of a bimodal contrast agent. Chem Eur J (in press, doi:10.1002/chem.201203773). Witte C, Kunth M, Döpfert J, Rossella F, Schröder L (2012) Hy-perpolarized xenon for NMR and MRI applications. J Vis Exp (67): e4268. Witte C, Schröder L (2012) NMR of hyperpolarised probes, NMR Biomed in press. (doi: 10.1002/nbm.2873) Schröder L, Faber C (2011) In vivo NMR imaging – Basic contrast mechanisms. Methods Mol Biol 771: 45-67. Ziegler A, Kunth M, Mueller S, Bock C, Pohmann R, Schröder L, Faber C, Giribet G (2011) Application of magnetic resonance im-aging in zoology. Zoomorphology 130: 227-254. FMP authorsGroup members

E X T E R N A L F U N D I N G European Union, 7. Framework Programme, ERC, “Biosensor-Imaging”: Hyperpolarized Biosensors in Molecular Imaging, ERC Starting Grant,12.2009 – 11.2014; 1.851.000 Euro Leibniz-Gemeinschaft, Leibniz Vorhaben im Rahmen des Pakts für Forschung und Innovation, „ Development of Novel NMR Pro-bes: Improving Cell Profiling for Early Diagnosis”, SAW , with C. Freund, 01.2011 – 12.2013; 761.613 Euro International Human Frontiers Science Program Organization, “Cell Profiling with Xenon Biosensors”, Long-Term Postdoctoral Fellowship for Christopher Witte, 04.2010 – 04.2013; 142.836 Euro

C O L L A B O R AT I O N S International Alexander ZieglerHarvard University, Cambridge, USA Alexander PinesLawrence Berkeley National Laboratory and University of California at Berkeley, USA

National Peter BachertDeutsches Krebsforschungszentrum, Heidelberg Cornelius FaberWestfälische Wilhelms-Universität Münster Christian FreundFreie Universität Berlin Ute Resch-GengerBundesanstalt für Materialforschung und -prüfung, Berlin Andreas HennigBundesanstalt für Materialforschung und -prüfung, Berlin Patrick StumpfFreie Universität Berlin Franz SchillingTechnische Universität München

Jörg Döpfert,

Martin Kunth

Federica Rossella,

Leif Schröder

Chris Witte,

Federica Rossella

Interaction of Cryptophanes with Lipid MembranesContrary to targeted molecular imaging applications, non-spe-cific interactions of xenon biosensors are poorly understood. Up-take of the hydrophobic xenon-binding moiety (a cryptophane cage) into the hydrophobic core of cell membranes was there-fore investigated. In collaboration with the Bundesanstalt für Materialforschung und -prüfung we developed a novel bi-modal contrast agent for NMR and fluorescence detection that allowed quantification of membrane association of cryptophane-dye con-jugates through FRET measurements and further characterization of changes in NMR signal contrast using the CEST approach.Selective readout of the caged xenon in different membrane en-vironments could be demonstrated as well as preliminary studies for a novel contrast mechanism based on the depolarization time of the CEST mechanism. This will enable agents that can sense membrane integrity and fluidity and thus reveal differences in micro-environments and their responses to drug delivery such as, for instance, the effect of antibiotics.

Novel Xenon BiosensorsAmong others, a new type for an enzyme-specific biosensor with a cleavage motif for MMP-11 (a marker for pancreatic cancer) is currently being developed. The enzymatic activity allows for converting substrate-labeled cryptophane cages into a modi-fied construct that shows a shift in the Xe NMR signal of ca. 1 ppm. The design of enzyme-responsive MR contrast agents has been the focus of recent research in many groups. Monitoring the catalytic activity of an enzyme, as opposed to stoichiometric protein binding, dramatically improves MR detection sensitivity. This is now, for the first time, paired with Hyper-CEST detection to further improve NMR sensitivity for disease-specific marker recognition.

58

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2

59

R E S E A R C H G R O U P S / / / S T R U C T U R A L B I O L O G Y F O R S C H U N G S G R U P P E N / / / S T R U K T U R B I O L O G I E

I N - C E L L N M R

N M R I N Z E L L E N

G R O U P L E A D E RD R . P H I L I P P S E L E N K O

B I O G R A P H Y

2002 Ph.D. at the European Molecular Biology Laboratory (EMBL), Heidelberg 2003 – 2008 Post-Doc at Harvard Medical School 2003 – 2004 EMBO fellowship 2004 – 2006 Human Frontiers in Science (HFSP) fellowship 2006 – 2007 Max Kade fellowship by the Austrian Academy of Science Since 2007 Group Leader in the Structural Biology Section of the FMP, Emmy Noether fellowship by the Deutsche Forschungsgemeinschaft (DFG)

S U M M A R Y

We employ high-resolution in-cell NMR spectroscopy to study the structural and func-tional properties of proteins inside living cells. We are particularly interested in biological processes that occur in higher eukaryotes and use model systems ranging from Xenopus laevis oocytes to cultured mammalian cells. Because NMR spectroscopy is an atomic-res-olution method that selectively detects isotope-labeled proteins in non isotope-labeled cellular environments, in-cell NMR permits a direct observation of proteins “in action” as they execute their biological functions. This tool can be compared to an “atomic resolu-tion microscope,” and we are using it for purposes such as the study of the early stages of neurodegenerative disease processes and initial events in aberrant cellular signaling pathways that lead to cancer. We are thereby gaining novel mechanistic insights into previously unknown cellular aspects of these diseases, opening new routes for drug dis-covery and therapeutic intervention.

Z U S A M M E N FA S S U N G

Mit hochauflösender In-Cell-NMR-Spektroskopie untersuchen wir die strukturellen und funktionalen Eigenschaften von Proteinen innerhalb von lebenden Zellen. Wir sind dabei besonders an den in höheren Eukaryoten stattfindenden biologischen Prozessen interes-siert und nutzen dafür Modellsysteme von Xenopus laevis-Oozyten bis hin zu Säugetier-Zellkulturen. Da die NMR-Spektroskopie als Methode mit atomarer Auflösung für eine selektive Detektion isotopenmarkierter Proteine in einer nicht mit Isotopen markierten zellulären Umgebung sorgt, erlaubt die In-Cell-NMR-Spektroskopie eine direkte Beob-achtung von Proteinen „in Aktion“, also während sie ihre biologischen Funktionen aus-üben. Sie kann mit einem „Mikroskop mit atomarer Auflösung“ verglichen werden und wir nutzen dies beispielsweise für das Studium von frühen Stadien neurodegenerativer Erkrankungen und anfänglichen Ereignissen abnormer zellulärer Signalwege, die zu Krebs führen. Wir gewinnen dabei neue mechanistische Einblicke in zuvor unbekannte zellu-läre Aspekte dieser Krankheiten und eröffnen so neue Wege für die Wirkstoffforschung und therapeutische Interventionen.

D E S C R I P T I O N O F P R O J E C T S

Using in-cell NMR spectroscopy, our laboratory explores physi-ological and pathological protein states in different cell types and under different cellular conditions. One of our major goals is to understand the structural in vivo properties of intrinsically disordered proteins (IDPs) and substantial efforts are dedicated to human alpha-synuclein, a neuronal IDP that is implicated in the development of Parkinson’s disease (PD). In PD patients, insoluble aggregates of alpha-synuclein deposit in dopaminergic neurons of the substantia nigra. These amyloid structures constitute patho-logical hallmarks of PD and result in the progressive loss of do-paminergic neurons. Using in-cell NMR spectroscopy we investi-gate how different cellular environments affect the conformational properties of alpha-synuclein and how they promote protein ag-gregation. We have recently completed the first atomic resolution analysis of human alpha-synuclein in five different mammalian cell types, which include cells of neuronal (3) and non-neuronal (2) origins, dopaminergic neurons (2) from the substantia nigra (1). We are now moving towards exposing these cells to conditions that promote alpha-synuclein aggregation and hope to thereby recapitulate early intracellular events in the course of Parkinson’s disease. To this end, we use pesticides, heavy metals, or reactive oxygen species (ROS), all of which have been shown to induce elevated levels of cellular oxidative stress and mitochondrial dys-function, which trigger intracellular amyloid formation. Our goal is to establish a cellular model system for Parkinson’s disease that we can study using in-cell NMR spectroscopy. Most eukaryotic proteins, including alpha-synuclein, are post-translationally modified and these modifications often confer important functional properties. We also employ in-cell NMR spectroscopy to directly monitor the establishment of cellular post-translational modifications and to analyze how these modi-fications affect a protein’s structure and function. Because in-cell NMR spectroscopy is a non-invasive and non-destructive method we study these cellular processes in a continuous, time-resolved and quantitative manner. This allows us to investigate the dynamic changes of cellular protein modification states in response to different signaling events. We are further developing new NMR methods to detect different types of cellular post-translational protein modifications. While protein phosphorylation and acety-lation events are of particular interest to us, we are also investi-gating cellular methylation, oxidation and proteolytic processing reactions.

We further employ in-cell NMR spectroscopy to study entire cel-lular signaling networks. Using peptide-based Kinase Activity Re-porters (KARs) and in-cell NMR readouts we ‘measure’ dynamic changes of cellular kinase activities under different physiologi-cal and pathophysiological conditions. KARs enable multiplexed readouts of cellular kinase activities in qualitative and quantitative terms. In addition, they allow us to monitor cellular kinase activi-ties and the dynamic changes thereof in real time. At present, we study global kinase activities in different human cancer cell lines and screen for drugs that selectively modulate individual signal-ing pathways. Besides protein phosphorylation and intracellular kinase activi-ties, we also design NMR reporters for acetyltransferases (HATs), deacetylases (HDACs), as well as methyltransferases (KMTs/PRMTs) and demethylases (DMs). All of our in-cell NMR reporters are built for multiplexed profiling purposes, which enable simultaneous NMR readouts of chemically distinct types of cellular PTMs in a single NMR experiment. In addition, we place a strong emphasis on the development of new in-cell NMR methods. We have de-veloped several protocols for the efficient delivery of recombinant proteins into a wide range of cultured mammalian cells. These approaches entail novel protein transduction techniques that can easily be applied to different proteins and different mammalian cell types.

Beata Bekei

60 61

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2

National Wolfgang FischleMax Planck Institute of Biophysical Chemistry, Göttingen Laura HartmannMax Planck Institute of Colloids and Interfaces, Berlin Remco SprangersMax Planck Institute for Developmental Biology, Tübingen Ulrich StelzlMax Planck Institute for Molecular Genetics, Berlin Erich WankerMax-Delbrück-Center for Molecular Medicine, Berlin Jan BieschkeMax-Delbrück-Center for Molecular Medicine, Berlin

R E S E A R C H G R O U P S / / / S T R U C T U R A L B I O L O G Y F O R S C H U N G S G R U P P E N / / / S T R U K T U R B I O L O G I E

G R O U P M E M B E R S Dr. Andres Binolfi * Dr. Stamatios Liokatis * Dr. Honor May Rose * Dr. Francois-Xavier Theillet * Beata Bekei (doctoral student) * Jonas Kosten (doctoral student) * Rossukon Thongwichian (doctoral student) * Silvia Verzini (doctoral student) * Sandy Goyette (technical assistant) * Marleen van Rossum (technical assistant) Marchel Stuiver (technical assistant)

Group members as of 31.12.2012 * Part of reporting period

Fig. 1: Overview of prokaryotic and eukaryotic

in-cell NMR systems. Protein over-expression

under isotope-labeled growth conditions of

bacterial cultures leads to the accumulation

of NMR-visible proteins, shown in red, in the

otherwise unlabeled environment of live cells

(top panel). Microinjection of isotope-labeled

proteins into Xenopus laevis oocytes enables the

efficient delivery of NMR-active biomolecules

into this type of eukaryotic cell (bottom panel,

left). Protein transduction into mammalian cells

can be achieved via tagging of isotope-labeled

proteins with cell-penetrating peptides, shown in

green (bottom panel, middle), or via pore-form-

ing bacterial toxins, also shown in green (bottom

panel, right).

Fig. 2: Establishing combinatorial post-transla-

tional protein modification (PTM) codes in chro-

matin. Dense nucleosomal arrays constitute the

basic building blocks of eukaryotic chromatin.

Core histones, the disc-shaped protein compo-

nents of chromatin assemble into nucleosomes,

which function as the molecular spools around

which all chromosomal DNA is wrapped in every

cell nucleus (left). Different combinations of

post-translational protein modifications at the

N-terminal ‘tails’ of histone proteins, shown here

for histone H3 in blue, establish the epigenetic

histone code that regulates the transcriptional

states of entire genomes (middle). Sets of sym-

metric and asymmetric histone PTMs, shown for

H3 as colored spheres, control gene activity in a

highly dynamic manner (right).

S E L E C T E D P U B L I C AT I O N S Binolfi A, Theillet FX, Selenko P (2012) Bacterial in-cell NMR of human alpha-synuclein: a disordered monomer by nature? Bio-chem Soc Trans 40: 950-954. Liokatis S, Stutzer A, Elsasser SJ, Theillet FX, Klingberg R, van Ros-sum B, Schwarzer D, Allis CD, Fischle W, Selenko P (2012) Phosphor-ylation of histone H3 Ser10 establishes a hierarchy for subsequent intramolecular modification events. Nat Struct Mol Biol 19: 819-823. Theillet FX, Liokatis S, Jost JO, Bekei B, Rose HM, Binolfi A, Schwarzer D, Selenko P (2012) Site-specific mapping and time-resolved monitoring of lysine methylation by high-resolution NMR spectroscopy. J Am Chem Soc 134: 7616-7619. Theillet FX, Smet-Nocca C, Liokatis S, Thongwichian R, Kosten J, Yoon MK, Kriwacki RW, Landrieu I, Lippens G, Selenko P (2012) Cell signaling, post-translational protein modifications and NMR spectroscopy. J Biomol NMR 54: 217-236. van Rossum B, Fischle W, Selenko P (2012) Asymmetrically modi-fied nucleosomes expand the histone code. Nat Struct Mol Biol 19: 1064-1066.

FMP authorsGroup members

E X T E R N A L F U N D I N G Deutsche Forschungsgemeinschaft, “The protein disorder para-dox: What do natively unfolded proteins look like inside living cells?”, SE 1794/1-1, I., 11.2007 – 10.2010 753.019 Euro Deutsche Forschungsgemeinschaft, “The protein disorder para-dox: What do natively unfolded proteins look like inside living cells?”, SE 1794/1-1, II. ,11.2010 – 02.2013, 260.000 Euro Deutsche Forschungsgemeinschaft, “The protein disorder para-dox: What do natively unfolded proteins look like inside living cells?” , SE 1794/1-1, III., 04.2012 – 06.2013, 242.000 Euro Leibniz-Gemeinschaft, Leibniz Vorhaben im Rahmen des Pakts für Forschung und Innovation, “In vivo NMR-Spektroskopie in humanen Zellen: Ein neues Instrument in der Systembiologie“, 01.2010 – 12.2012, 761.000 Euro

C O L L A B O R AT I O N S International Axel BehrensCancer Research UK, London, United Kingdom Donal O’CarrollEuropean Molecular Biology Laboratory (EMBL), Rome, Italy Gary DaughdrillUniversity of South Florida, USA Isabella FelliCenter for Magnetic Resonance (CERM), Florence, Italy Stephan GrieszekBiozentrum Basel, Switzerland Kyou-Hoon HanKorea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Korea Guy LippensUniversity of Lille, France Roberta PieratelliCenter for Magnetic Resonance (CERM), Florence, Italy Peter TompaHungarian Academy of Science and University of Brussels, Belgium Valdimir UverskyUniversity of South Florida, USA

Rossukon (Tim) Thongwichian,

Andreas Binolfi

Jonas Kosten,

François-Xavier Theillet

Marchel Stuiver

R E S E A R C H H I G H L I G H T S / / / N M R F Ü R G A N Z E U R O PA

62 63

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2

MOLECULAR PHYSIOLOGY AND CELL BIOLOGY SECTION

BEREICH MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE

Behavioural NeurodynamicsVerhaltensneurodynamik

GROUP LEADERS Dr. Tatiana Korotkova / Dr. Alexey Ponomarenko

PAGE 78

Molecular Neuroscience and BiophysicsMolekulare Neurowissenschaften und Biophysik

GROUP LEADER Dr. Andrew J. R. Plested

PAGE 82

Protein Trafficking

GROUP LEADER PD Dr. Ralf Schülein

PAGE 86

Cellular Imaging / Electron MicroscopyZelluläre Bildgebung / Elektronenmikroskopie

GROUP LEADER Dr. Burkhard Wiesner

PAGE 90

Physiology and Pathology of Ion TransportPhysiologie und Pathologie des Ionentransports

GROUP LEADER Prof. Dr. Dr. Thomas J. Jentsch

PAGE 66

Molecular Cell PhysiologyMolekulare Zellphysiologie

GROUP LEADER PD Dr. Ingolf E. Blasig

PAGE 70

Molecular Pharmacology and Cell BiologyMolekulare Pharmakologie und Zellbiologie

GROUP LEADER Prof. Dr. Volker Haucke

PAGE 74

64 65

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2M O L E C U L A R P H Y S I O L O G Y A N D C E L L B I O L O G Y S E C T I O N B E R E I C H M O L E K U L A R E P H Y S I O L O G I E U N D Z E L L B I O L O G I E

The most important development for the section has been the establishment of the Department of “Molecular Pharmacology and Cell Biology” headed by Volker Haucke, who joined the FMP as a new Director in 2012 and profoundly changed the sections research portfolio; together with the department’s subgroups led by Michael Krauß, Tanja Maritzen, and Jan Schmoranzer, he sig-nificantly boosts the section focus on neurobiology and endocytic membrane trafficking, with a particular emphasis on the synapse, and importantly strengthens the biochemical expertise of the FMP. With the identification of Pitstops, potent inhibitors of endocyto-sis, his research recently provided an excellent example for drug screening resulting in important cell biological tools. Concerning research infrastructure, his department furthermore introduces state-of-the-art super-resolution light microscopy together with Jan Schmoranzer. This has led to a collaborative effort to build a strong molecular imaging platform jointly with the core facility

“Cellular Imaging” headed by Burkhard Wiesner, which provides a whole repertoire of light microscopic and electron microscopic methods complementary to super-resolution light microscopy.

The latest development is the hiring of another Neurocure-asso-ciated Junior Group, “Cellular Proteostasis”, focussed on protein dynamics in the model organism C. elegans led by Janine Kirstein-Miles, who will commence work at the FMP in September 2013. Research of the section is well interconnected with research within the other sections of the FMP, in particular with the screening and chemical biology capacities provided by the “Screening Unit” and the “Chemical Biology Unit” as well as with the proteomics capabilities of the “Mass Spectrometry” group of the Chemical Biology section. Overall, the whole spectrum of research within the section significantly bolsters our focus on neurobiology and cell biology, and provides an excellent basis for future research into crucial cellular mechanisms that may be amenable to phar-macological intervention.

von Tight-Junction-Molekülen erfolgreich fort. Diese Proteine haben wichtige Funktionen bei der Etablierung und Aufrecht-erhaltung organischer Barrieren wie die der Blut-Hirn-Schranke. Ralf Schülein und seine Arbeitsgruppe „Protein-Trafficking“ un-tersuchen die Biogenese und Funktion von G-Protein-gekoppel-ten Rezeptoren, v.a. von den Rezeptoren, die an der Stressant-wort des Körpers beteiligt sind. So konnte kürzlich gezeigt werden, dass der Corticotropin-Releasing-Factor (CRF)-Rezeptor 2a im Ge-gensatz zu den meisten anderen GPCRs in der Plasmamembran als Monomer vorliegt.

Das bedeutendste Ereignis in diesem Bereich war die Einrich-tung der von Volker Haucke geleiteten neuen Abteilung „Mole-kulare Pharmakologie und Zellbiologie”, welche ferner drei Unter-gruppen beherbergt, die von Michael Krauß, Tanja Maritzen und Jan Schmoranzer geführt werden. Der wissenschaftliche Fokus von Volker Haucke liegt auf der molekularen Neurobiologie - mit einem Hauptaugenmerk auf der Synapse - sowie auf den Mecha-nismen des endozytotischen Membrantransports. Ferner stärkt seine Abteilung maßgeblich die biochemische Kompetenz des gesamten Instituts. Mit der Identifizierung von „Pitstops“, wirk-samen Endozytoseinhibitoren, lieferte die Arbeitsgruppe kürz-lich ein ausgezeichnetes Beispiel dafür, wie das Wirkstoff-Scree-ning zu wichtigen zellbiologischen Werkzeugen führen kann. Im Bereich der Forschungsinfrastruktur des Instituts führte das Team um Jan Schmoranzer Methoden für die höchstauflösende Lichtmi-kroskopie ein, die an der Spitze der technologischen Entwicklung stehen. In enger Zusammenarbeit mit der von Burkhard Wiesner geleiteten Arbeitsgruppe „Zelluläre Bildgebung“ entsteht somit eine moderne Technologie-Plattform, die das Repertoire lichtmi-kroskopischer und elektronenmikroskopischer Methoden in gan-zer Breite zur Verfügung stellt.

Das Ende des Berichtzeitraums krönte die erfolgreiche Etablie-rung der ebenfalls mit NeuroCure assoziierten Nachwuchsgruppe

„Zelluläre Proteostase“. Die neue Gruppenleiterin, Janine Kirstein-Miles, nimmt ihre Tätigkeit im September 2013 auf und wird die Proteindynamik im Modellorganismus C. elegans untersuchen. Zusammengefasst erfuhr der Bereich eine deutliche Stärkung seiner neurobiologischen und zellbiologischen Kompetenz. Das Forschungsspektrum bietet jetzt insgesamt eine ausgezeichnete Basis für die Untersuchung zellulärer Mechanismen, die pharma-kologisch beeinflusst werden können.

Life is based on complex cellular and physiological mechanisms and their well-orchestrated interplay. In disease, this interplay gets out of balance. Research in the section “Molecular Physiol-ogy and Cell Biology” aims at understanding such mechanisms in molecular detail, as well as their dysfunction in disease. Cellular targets for pharmaceutical intervention, many of them membrane proteins, and prominently placed in this subset ion channels and G-protein coupled receptors, are identified, studied in their physi-ological environment, and their modulation by bioactive com-pounds is explored. According to our mission to create a broader basis for pharmacology we focus on the study of less explored membrane proteins and of molecules of key importance for intra-cellular trafficking. To this end, we capitalize on a broad arsenal of techniques, ranging from molecular and cellular biology over biochemistry and biophysics to whole-animal physiology using genetically modified mice, often with conceptual links to human disease. Our projects have benefitted greatly from interactions with other sections of the FMP, including those concerned with structural biology and modeling, drug and siRNA screening, as well as chemical biology.

Two main topics addressed in this section concern membrane proteins like ion channels and transporters, receptors, and junc-tional proteins, as well as cellular trafficking processes, in particular endo- and exocytosis. Strong links exist between these two top-ics: for instance, the interplay between exocytic membrane inser-tion of receptors into the plasma membrane and their endocytic retrieval determines the cellular activity of receptors; conversely, ion transport across endo-/lysosomal membranes regulates intra-cellular trafficking.

During the reporting period, research within this section has sig-nificantly progressed due to exciting discoveries made by groups present during the entire reporting period. The Department of

“Physiology and Pathology of Ion Transport” headed by Thomas Jentsch made seminal contributions to the role of ion transport in endosomes and lysosomes, expanded its research to new ion channel classes and established a focus on sensory biology. An-drew Plested, leader of the Junior Group “Molecular Neurosci-ences and Biophysics” and member of the NeuroCure Cluster of Excellence, has continued his exciting work on the structure-function-relationship of postsynaptic glutamate receptors that play crucial roles in learning and memory. The Junior Group “Be-havioural Neurodynamics”, headed by Alexei Ponomarenko and Tatjana Korotkova, has been co-appointed with Neurocure and has been partially funded by an SAW-grant to Thomas Jentsch on KCNQ channels. It is located at the Neurocure building in Charité Mitte and importantly fills the gap of in vivo electrophysiologi-cal recordings from mouse models, including those for KCNQ K+ channels. The “Molecular Cell Physiology” group headed by Ingolf Blasig continued its in-depth investigation of tight junc-tion molecules with important implications for organ barriers like the blood-brain barrier. Ralf Schülein and his Protein-Trafficking group explore G-protein coupled receptor biogenesis and func-tion. The Schülein group recently reported the important result that corticotropin-releasing factor receptor 2a, in contrast to most other GPCRs, functions as a monomer.

Die Grundlage einer funktionierenden Zelle ist ein perfektes Zu-sammenspiel einzelner Moleküle. Ist dieses Gleichgewicht ge-stört, können Krankheiten entstehen. Die Wissenschaftler des Be-reichs „Molekulare Physiologie und Zellbiologie” untersuchen die zugrunde liegenden komplexen zellulären und physiologischen Mechanismen sowie deren krankheitsbedingte Funktionsstörun-gen. Einige der Forschergruppen definieren pharmakologisch re-levante Zielstrukturen an Zellen. Dabei handelt es sich meist um Membranproteine, insbesondere um Ionenkanäle und G-Protein-gekoppelte Rezeptoren (GPCR). Entsprechend dem Auftrag des Instituts, in der Zukunft die Arzneimitteltherapie auf eine breitere Basis zu stellen, konzentrieren sich diese Untersuchungen auf bis-her wenig erforschte Membranproteine und Moleküle. Das Inter-esse liegt dabei nicht nur auf der physiologischen Umgebung der Zielstrukturen, sondern auch auf der Beeinflussung der Proteine durch Wirkstoffe. Die Arbeitsgruppen wenden hierfür ein breites Spektrum an Methoden an – von Molekular- und Zellbiologie über Biochemie und Biophysik bis hin zu Analysen am gesamten tieri-schen Organismus. Für Letzteres setzen die Forscher in der Regel genetisch modifizierte Mäuse ein, die oftmals auch Modelle für humane Erkrankungen darstellen.

Neben Membranproteinen bilden zelluläre Transportprozesse einen weiteren Fokus, wobei die beiden Schwerpunkte eng mit- einander verknüpft sind: So bestimmt das Zusammenspiel zwi-schen dem Einbau von Rezeptoren in die Plasmamembran (Exo-cytose) und ihrer Wiederaufnahme in die Zelle (Endocytose) die zelluläre Aktivität von Rezeptoren; umgekehrt reguliert der Ionen-transport über endo-/lysosomale Membranen den intrazellulären Transport.

Während des Berichtszeitraums erzielten die Arbeitsgruppen Auf-sehen erregende Forschungsergebnisse, wobei sie oftmals auch von Kooperationen mit Forschungsgruppen anderer Bereiche des Instituts profitierten. Die von Thomas Jentsch geleitete Ab-teilung „Physiologie und Pathologie des Ionentransports” lie-ferte wegweisende Beiträge zur Rolle des Ionentransports in Endosomen und Lysosomen, dehnte ihre Forschung auf neue Ionenkanal-Klassen aus und entwickelte einen neuen Schwer-punkt im Bereich der molekularen Mechanismen der Sensorik. Andrew Plested, Leiter der Nachwuchsgruppe „Molekulare Neu-rowissenschaft und Biophysik” und Mitglied im Exzellenzclus-ter NeuroCure, hat seine Arbeiten zu Struktur-Funktionsbezie-hungen postsynaptischer Glutamat-Rezeptoren sehr erfolgreich fortgeführt. Diese Rezeptoren spielen nicht nur eine entschei-dende Rolle beim Lernen und Erinnerungsvermögen, sondern auch bei bestimmten Formen der Epilepsie. Die Nachwuchs-gruppe „Verhaltensneurodynamik” von Alexey Ponomarenko und Tatiana Korotkova wurde in das Exzellenzcluster Neuro-Cure aufgenommen und durch SAW-Mittel gefördert, die Tho-mas Jentsch für Arbeiten an KCNQ-Kanälen eingeworben hatte. Die Gruppe ist im NeuroCure-Gebäude an der Charité-Uni-versitätsmedizin Berlin-Mitte angesiedelt und benutzt elektro-physiologische in vivo Untersuchungen an Mäusen, die z.B. als Modelle für KCNQ-Kaliumkanäle fungieren und so eine wich-tige Lücke am FMP füllen. Die Arbeitsgruppe „Molekulare Zell-physiologie“ von Ingolf Blasig setzte ihre Untersuchungen

M O L E C U L A R P H Y S I O L O G Y A N D C E L L B I O L O G Y S E C T I O N B E R E I C H M O L E K U L A R E P H Y S I O L O G I E U N D Z E L L B I O L O G I E

66 67

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2R E S E A R C H G R O U P S / / / M O L E C U L A R P H Y S I O L O G Y A N D C E L L B I O L O G Y F O R S C H U N G S G R U P P E N / / / M O L E K U L A R E P H Y S I O L O G I E U N D Z E L L B I O L O G I E

P H Y S I O L O G Y A N D PAT HOLOGY OF IO N T RA N S P O R T

P H Y S I O L O G I E U N D PAT H O L O G I E D E S I O N E N T R A N S P O R T S

GROUP LEADERPROF. DR. DR. THOMAS J. JENTSCH

B I O G R A P H Y

1972 – 1978 Studied medicine at the FU Berlin

1974 – 1980 Studied physics at the FU Berlin

1981 – 1985 Staff scientist at the Institut für Klinische Physiologie (Prof. Wiederholt), FU Berlin

1982 Ph.D. in physics at the Fritz- Haber-Institute (Prof. Block), Berlin

1984 M.D. at the Institut für Klinische Physiologie (Prof. Wiederholt), FU Berlin

1986 – 1988 Postdoctoral fellow at the Whitehead Institute (Harvey F. Lodish, MIT), Cambridge MA

1988 – 1993 Research group leader at the Centre for Molecular Neurobiology Hamburg (ZMNH), Hamburg University

1991 “Habilitation” in Cell Biochemistry at the Medical School of Hamburg University

1993 – 2006 Full professor (C4) of Molecular Neuropathology at the ZMNH, Hamburg University; Director of the Institut für Molekulare Neuropathobiologie

1995 – 1998 and 2001 – 2003 Director of the Centre for Molecular Neurobiology Hamburg (ZMNH)

since 2006 Head of department at FMP and MDC, Berlin (joint appointment), Full Professor (W3) at Charité – Universitäts-medizin Berlin

since 2007 Member of NeuroCure Cluster of Excellence

since 2009 Deputy Director of Leibniz- Institut für Molekulare Pharmakologie (FMP)

S U M M A R Y

We aim to understand ion transport processes from the molecular (structure-function analysis) to the subcellular and cellular levels, up to the level of the organism. The latter levels are addressed through an investigation of the phenotypes of knock-out (KO) and knock-in (KI) mice and the analysis of human genetic diseases. We focus on CLC chloride channels and transporters, KCNQ potassium channels, KCC cation-chloride cotrans-porters, and Anoctamin Ca2+-activated chloride channels. Important research areas are the endosomal/lysosomal system and the control of neuronal excitability. We study many organs, including the brain, inner ear, olfactory epithelium, skin mechanoreceptors, kid-ney, intestine, and bone.

Z U S A M M E N FA S S U N G

Unser Ziel ist es, Ionentransportprozesse auf molekularer Ebene (Struktur-Funktionsana-lyse) über subzelluläre und zelluläre Prozesse bis hin zum Gesamtorganismus zu verste-hen. Letzteres versuchen wir durch Untersuchung der Phänotypen von knock-out (KO)- und knock-in (KI)-Mäusen und die Analyse humangenetischer Erkrankungen zu erreichen. Wir konzentrieren uns dabei auf CLC-Chloridkanäle und -Transporter, KCNQ-Kaliumka-näle, KCC-Kation-Chlorid-Cotransporter und Anoctamin Ca2+-aktivierte Chloridkanäle. Wichtige Forschungsbereiche sind das endosomal/lysosomale System und die Steue-rung neuronaler Erregbarkeit. Wir untersuchen viele Organe, u.a. Gehirn, Innenohr, Riech-epithel, Mechanorezeptoren der Haut, Niere, Darm und Knochen.

D E S C R I P T I O N O F P R O J E C T S

CLC chloride channels and transporters Proteins of the CLC gene family, discovered by us in 1990, reside in the plasma membrane and intracellular vesicles. We have gen-erated KO mouse models for most CLCs and have identified cor-responding human diseases, yielding insights into their diverse physiological roles. We have also identified ancillary b-subunits. Mutations in their genes also cause human disease. Surprisingly, vesicular CLCs are Cl-/H+-exchangers, suggesting that they have functions beyond acidification of intracellular vesicles. This was recently confirmed by mouse models in which we converted ClC-5 and ClC-7 into pure Cl- conductors. We are now analyzing similar models for ClC-3 and are generating new vesicular CLC mouse models carrying other interesting point mutations to clarify spe-cific roles in synaptic vesicles and in endo-lysosomal trafficking. Using mutations in sorting signals, we misdirected a portion of the lysosomal ClC-7 to the cell surface and showed that it opens only very slowly upon depolarization. Surprisingly, several human os-teopetrosis-causing mutations accelerated ClC-7 gating. We now investigate whether this change in kinetics is pathogenic per se.We have previously shown that disruption of the plasma mem-brane Cl- channel ClC-2 leads to leukodystrophy in mice. We did not find ClC-2 mutations in patients with leukodystrophy, but recent work (with R. Estévez and M. Pusch) shows that GlialCAM, a cell adhesion molecule underlying a form of human leukodys-trophy, associates with ClC-2 and changes its properties. We are now investigating whether changes in ClC-2 underlie the glial pathology caused by GlialCAM mutations. Anoctamin (TMEM16) Ca2+-activated chloride channelsAnoctamins form a recently identified family of Ca2+-activated Cl- channels. We have shown that Ano2 is the long-sought Ca2+-activated Cl- channel of olfactory sensory neurons, which was supposed to be crucial in amplifying olfactory responses. Surpris-ingly, however, our Ano2-/- mice showed that Ano2 is dispensable for olfaction. Whereas the main olfactory epithelium expresses only Ano2, the vomeronasal organ (VNO), which is specialized in detecting odors relevant for social interactions, also expresses Ano1. We are now studying conditional double KOs of both Ca2+ activated Cl- channels in the VNO.

KCNQ potassium channels We previously cloned and characterized the K+ channels KCNQ2-5, showed that mutations in KCNQ2 and 3 cause neonatal epi-lepsy, and mutations in KCNQ4 DFNA2-type dominant deafness. KCNQ2-5 mediate M-type currents that regulate neuronal excit-ability. We have recently generated mouse models for KCNQ4 and KCNQ5. Both channels are expressed in the vestibular organ, but unlike KCNQ4 in the cochlea, they do not localize to sen-sory hair cells but rather to postsynaptic membranes of afferent vestibular neurons. Functional analysis of our KCNQ4 KO mice revealed a mild vestibular phenotype that is relevant for DFNA2 patients. We discovered that KCNQ4 is also expressed in certain skin mechanoreceptors. In collaboration with G. Lewin, we found that the tuning of these mechanoreceptors is impaired in our KCNQ4 mouse models. Patients with KCNQ4-related hearing loss displayed a similar change in touch sensation. Potassium-chloride cotransporters We have previously generated and analyzed constitutive KOs of the KCC K+-Cl--cotransporters KCC1– KCC4 and discovered un-expected functions in various tissues. The neuronal isoform KCC2 lowers intraneuronal Cl- concentration, a process required for the inhibitory response to the neurotransmitters GABA and glycine. We are currently investigating the role of KCC2 in defined neuron populations using cell-type specific deletions. In recently pub-lished work, we investigated synaptic inhibition in cerebellar neu-ronal circuits in mice with granule and Purkinje-cell specific KCC2 disruption. Intracellular Cl- was increased in both Purkinje- and granule cells, confirming the role of KCC2 as the major neuronal Cl- extruder. Owing to a resting Cl- conductance, granule cells were depolarized and hyperexcitable when lacking KCC2, in turn leading to impaired cerebellar vestibulo-ocular learning. Analysis of mouse models with other cell-type specific KCC2 deletion is in progress.

Jonas Münch,

Ian Orozco

Felizia Voß,

Tobias Stauber

68 69

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S G R U P P E N / / / M O L E K U L A R E P H Y S I O L O G I E U N D Z E L L B I O L O G I E

S E L E C T E D P U B L I C AT I O N S Seja P, Schonewille M, Spitzmaul G, Badura A, Klein I, Rudhard Y, Wisden W, Hübner CA, de Zeuw CI, Jentsch TJ (2012). Raising cytosolic Cl- in cerebellar granule cells affects their excitability and vestibulo-ocular learning. EMBO J 31: 1217-1230. Spitzmaul G, Tolosa L, Winkelman BHJ, Heidenreich M, Frens MA, Chabbert C, de Zeeuw CI, Jentsch TJ (2013). Vestibular role of KCNQ4 and KCNQ5 K+ channels revealed by mouse models. JBC 288 (13), 9334-9344. Heidenreich M, Lechner SG, Vardanyan V, Wetzel C, Cremers CW, De Lenheer EM, Aránguez G, Moreno-Pelayo MA, Jentsch TJ*, Lewin GR* (2012). KCNQ4 K+ channels tune mechanorecep-tors for normal touch sensation in mouse and man. Nat Neurosci 15: 138-145. Billig GM, Pál B, Fidzinski P, Jentsch TJ (2011). Ca2+-activated Cl-

channels are dispensable for olfaction. Nat Neurosci 14: 763-749. Leisle L, Ludwig CF, Wagner FA, Jentsch TJ*, Stauber T (2011). ClC-7 is a slowly voltage-gated 2Cl-/H+ exchanger and requires Ostm1 for transport activity. EMBO J 30: 2140-2152. FMP authors Group members * corresponding authors

E X T E R N A L F U N D I N G Deutsche Forschungsgemeinschaft, “Ionentransporter und GA-BAerge Transmission“, JE 164/8-1, 07.2008 – 06.2011, 491.895 Euro Deutsche Forschungsgemeinschaft, SFB 740, C05, “Protein mo-dules involved in vesicular acidification and trafficking: focus of CIC-6”, 01.2007-12.2010. continued as: SFB 740/2, C05, 01.2011 – 12.2014, 688.000 Euro Deutsche Forschungsgemeinschaft, “Der CIC-7/Ostm1 Chlo-ridtransporter in Lysosomen und Osteoklasten“, JE 164/7-1, 01.2007 – 08.2010, continued as: JE 164/7-2, 07.2011 – 07.2014, 669.000 Euro Deutsche Forschungsgemeinschaft, “Strukturelle Grundlagen und physiologische Funktion des Cl-/H+-Gegenaustausches bestimmter CLC-Chloridtransportproteine“, ZD 58/1-1, with A. Zdebik, 07.2006 – 10.2010, continued as: JE 164/9-2, 01.2011 – 12.2013, 320.500 Euro Leibniz-Gemeinschaft, “Leibniz-Vorhaben im Rahmen des ‘Pak-tes für Forschung und Innovation‘“, 01.2009 – 12.2011, 384.000 Euro Prix Louis Jeantet, 04.2000 – 12.2012, 308.008 Euro Europäischer Forschungsrat (7. Forschungsrahmenprogramm), „Ion homeostasis and volume regulation of cells and organelles (CYTOVOLION)“, ERC # 294435, 04.2012 – 04.2017, 2.096.800 Euro Deutsche Forschungsgemeinschaft, Exzellenzinitiative an der Humboldt-Universität zu Berlin, Projekt NeuroCure: “Towards a better outcome of central nervous system disorders”, 01.2008 – 12.2011, 120.000 Euro MDC-Graduate-School, PhD grants to Sebastian Schütze, Till Stuhlmann, Felizia Voß Leibniz-Graduate-School of Biophysics, PhD grant to Carmen Ludwig

R E S E A R C H G R O U P S / / / M O L E C U L A R P H Y S I O L O G Y A N D C E L L B I O L O G Y

G R O U P M E M B E R S Dr. Luiza Bengtsson * Dr. Gwendolyn Billig Dr. Pawel Fidzinski * Dr. Chandresh Gajera * Dr. Maja Hoegg-Beiler * Dr. Sabrina Jabs Dr. Hermann-Josef Kaiser * Dr. Lilia Leisle * Dr. Matthias Heidenreich * Dr. Ian Orozco *Dr. Balazcs Pál * Dr. Rosa Planells-Cases (visiting scientist) * Dr. Guillermo Spitzmaul * Dr. Tobias Stauber

Dr. Stefanie Weinert Dr. Dietmar Zimmer (research coordinator) *Norma Nitschke (research coordinator) * Sebastian Albrecht (doctoral student) * Eun-Yeong Bergsdorf (doctoral student) * Andreia Cruz e Silva (doctoral student) * Kathrin Gödde (doctoral student) Carmen Ludwig (doctoral student) Jonas Münch (doctoral student) * Karina Oberheide (doctoral student) Sebastian Schütze (doctoral student) Kristin Schnuppe (doctoral student) Patricia Seja (doctoral student) * Till Stuhlmann (doctoral student) * Florian Ullrich (doctoral student) *

Felizia Voß (doctoral student) Carolin Backhaus (technical assistant) * Anyess von Bock (technical assistant) Petra Göritz (animal care taker) Nicole Krönke (technical assistant) Rainer Leben (technical assistant) * Janet Liebold (technical assistant) Ruth Pareja (technical assistant) Katrin Räbel (technical assistant) Mario Ringler (lab coordinator) * Patrick Seidler (technical assistant) Andrea Weidlich (technical assistant) * Silke Zillmann (technical assistant) * Group members as of 31.12.2012 * Part of period reported

Fig. 1: Immunohistochemistry of neurons at the base of a hair shaft (blue).

Myelinated mechanosensitive neurons are stained in green for neurofilament

200. Surrounding the hair shaft, they express the KCNQ4 K+ channel (red)

at sites that are important for transducing mechanical forces into electrical

signals. (see Heidenreich et al. (2012) Nature Neuroscience 15, 138-145)

Fig. 2: Immunohistochemistry of neurons in the cerebellum. Green staining

shows the localization of the potassium-chloride cotransporter 2 in different

neuron types that control motorcoordination. In red: Purkinje-cells stained

with the marker Parvalbumin. (see Seja et al. (2012) EMBO J 31, 1217-1230)

C O L L A B O R AT I O N S International Gracia AránguezHospital General Universitario Gregorio Marañón, Madrid, Spain Andrea BallabioTelethon, Napoli, Italy, and Texas Children’s Hospital, Houston, USA Christian ChabbertInstitut de Neurosciences de Montpellier, France Carole CharlierUniversité de Liège, Belgium Jonathan D. CooperKing’s College, London, GB Cor CremersRadboud University Nijmegen Medical Centre, The Netherlands Dominique EladariHôpital Européen Georges Pompidou, Paris, France Raúl EstévezUniversity of Barcelona, Spain Igor MedinaINMED/ INSERM, Marseilles, France Miguel Ángel Moreno-PelayoHospital Universitario Ramón y Cajal, Madrid, Spain Kyoshi MoriKyoto University, Japan

Michael PuschCNR, Genova, Italy Francisco SepúlvedaCECS, Valdivia, Chile Richard SmithUniversity of Iowa, Iowa City, USA Jana TyyneläUniversity of Helsinki, Finland Shinichi UchidaThe University of Tokyo, Japan Marjo Van der KnaapNeuroscience Campus Amsterdam, The Netherlands Chris de ZeeuwErasmus MC, Rotterdam and Netherlands Institute for Neuroscience, Amsterdam, The Netherlands

National Bernd FaklerUniversity of Freiburg Maik GollaschCharité – Universitätsmedizin Berlin Christian HübnerUniversitätsklinikum Jena Uwe KornakCharité – Universitätsmedizin Berlin and Max-Planck-Institute for Molecular Genetics, Berlin Jens von KriesLeibniz-Institut für Molekulare Pharmakologie (FMP), Berlin Gary LewinMax-Delbrück-Center for Molecular Medicine, Berlin Hannes MaierMedizinische Hochschule Hannover Alexey PonomarenkoCharité – Universitätsmedizin Berlin and FMP, Berlin Dietmar SchmitzCharité – Universitätsmedizin Berlin Björn SchroederMax-Delbrück-Center for Molecular Medicine, Berlin Frank ZufallUniversität des Saarlandes, Homburg/Saar

Matthias Heidenreich

Stephanie Wernick,

Nicole Krönke,

Anyess von Bock

70 71

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2R E S E A R C H G R O U P S / / / M O L E C U L A R P H Y S I O L O G Y A N D C E L L B I O L O G Y F O R S C H U N G S G R U P P E N / / / M O L E K U L A R E P H Y S I O L O G I E U N D Z E L L B I O L O G I E

M O L E C U L A R C E L L P H Y S I O L O G Y

M O L E K U L A R E Z E L L P H Y S I O L O G I E

G R O U P L E A D E RP D D R . I N G O L F E . B L A S I G

B I O G R A P H Y

1970 – 1974 Studied biology and bio- chemistry in Leipzig, diploma thesis on cancer research at the Robert-Rössle- Hospital in Berlin-Buch

1984 Dissertation on the pharmacology of myocardial infarction at the Academy of Sciences, Berlin

1992 Habilitation for investigations on reactive species in myocardial dysfunction and venia legendi for biochemical pharma-cology, University of Halle-Wittenberg

since 1992 Head of the independent re-search group for Molecular Cell Physiology at the FMP, teaching of pharmacology, functional biochemistry, neurochemistry at the universities in Potsdam and Berlin

1993 – 1995 Awarded project leader at the NIH, Washington, DC/USA

S U M M A R Y

We focus on the structure, function, and modulation of cell-cell contacts to explore tight junctions (TJ) in tissue barriers, in order to disclose pathological mechanisms that might lead to improved therapies. One outcome of this work is to propose new strategies that specifically manipulate neurological and other barriers to improve drug delivery and to prevent barrier dysfunction.The paracellular tightness of epithelia and endothelia is determined by membrane proteins: claudins, tight junction-associated marvel proteins, TAMPs, (occludin, tricellulin, mar-velD3) and associated scaffolding proteins (e.g. ZO-1). It is unclear how the structure, regulation, and interactions of these proteins lead to opening or tightening of TJs. Our work focuses on processes that permit the oligomerization of key proteins of tissue barri-ers and mechanisms to open and/or to reconstitute the TJ after opening.Every year we organize an international symposium to disseminate news of progress in the field and thus stimulate collaborations, publications, patents and new funding.

Z U S A M M E N FA S S U N G

Wir beschäftigen uns mit der Struktur, Funktion und Modulierung von Zell-Zellkontak-ten und erforschen die „Tight Junctions“ (TJs, permeationsdichte Zellzwischenräume) an Gewebsgrenzen, um pathologische Mechanismen aufzudecken, die dann langfristig zu besseren Therapien führen sollen. Ein Forschungsziel besteht darin, neue Strategien vorzuschlagen, mit denen man neurologische und andere Barrieren gezielt manipulie-ren kann, um den Wirkstofftransport zu verbessern und Barrierestörungen zu verhindern.Die parazelluläre Dichtigkeit von Epithelien und Endothelien wird durch Membranprote-ine bestimmt: Claudine, tight junction-associated marvel proteins, TAMPs, (Occludin, Tri-cellulin, MarvelD3) und assoziierte Gerüstproteine (z.B. ZO-1). Noch ist unklar, wie Struk-tur, Regulationsmechanismen und Wechselwirkungen dieser Proteine zur Öffnung bzw. Schließung der TJs führen. Wir konzentrieren uns auf Prozesse, die die Oligomerisierung von Schlüsselproteinen der Gewebsbarrieren erlauben und Mechanismen, die die TJs öffnen und/oder nach Öffnung rekonstituieren.Zur Verbreitung der auf diesem Forschungsgebiet erzielten Fortschritte organisieren wir jährlich ein internationales Symposium und regen so Kooperationen, Publikationen, Ent-wicklung von Patenten sowie die Einwerbung neuer Fördermittel an.

D E S C R I P T I O N O F P R O J E C T S

Elucidation of the tightening mechanism of the tight junctions:We systematically analyzed the interaction potential between the main TJ proteins (Fig. 1, p. 72). These studies demonstrated that the majority of the members of the highly ho-mologous classic claudins are able to associate homo- and heterophilically. In particular, the extracellular loops were involved in the associations (Piontek et al., 2011). Similarly, but to a lesser degree, the TAMPs interact homo- and heterophilically – apart from oc-cludin and tricellulin, which cannot bind to each other. For the first time, direct binding of TAMPs with classic claudins was demonstrated (Cording et al., 2012). These interactions showed that claudins may determine binding properties, membrane localization or mo-bility of the TAMPs and, conversely, TAMPs determine the strand morphology of the clau-

dins. In general, the interactions found between the TJ proteins provide deeper insights into the TJ assembly (Blasig and Haseloff, 2011). Claudin-1 and claudin-5 were found as preferred interaction partners with the other constituents of the TJ. As claudin-5 is es-sential for the tightness of the blood-brain barrier and claudin-1 for the perineurium ensheathing peripherial nerves, both claudins were established as new pharmacological and diagnostic targets.

New regulatory role of tight junction proteins:The molecular function of the TAMPs is not known. For occludin, a unique TJ marker, we developed the new concept that redox-dependent signal transduction mechanisms and redox-sensitive domains of occludin play a key role in the redox regulation of pro-tein interactions in the TJs, which has a major impact in diseases related to oxidative stress and corresponding pharmacological interventions (Blasig et al., 2011). Moreover, the oligomerization status of occludin influenced its association with other TJ pro-teins, e.g. claudin-5 and ZO-1. Further investigations showed that redox-sensitive cysteines in the extracellular loops of occludin, tricellulin and marvelD3 are involved in the interactions observed. ZO-1, the scaffolding protein of the TJs, was found to be affected by regulatory proteins. We identified the SH3-hinge-GuK unit in ZO-1 as a multiple binding site for a number of signal transduction elements. In addition, this unit was able to oligomerize (Rückert et al., 2012). In conclusion, proteins with different regulatory po-tential, e.g. G-proteins, adhere to and influence cellular functions of ZO proteins. Moreover, the interactions can be modulated, e.g. by phosphorylation of the hinge region and/or activation of the binding proteins (Bal et al., 2012).

New modulators of tight junctions: Claudins form the backbone of the TJ strands, and their composi-tion determines the barrier function in a tissue. Modulation of such barriers is therefore of potential pharmaceutical interest. The first extracellular loop of claudin-1 was noticed to be involved in the interaction between TJ proteins. Therefore, peptidomimetics of this loop with ß-sheet potential were generated and transiently increased the paracellular permeability for ions, high and low mo-lecular weight compounds in cell models (Fig. 2, p. 72). Perineu-rial injection in rats facilitated the uptake of anesthetics into the nerve (Zwanziger et al., 2012a). The mechanism is related to the internalization of the peptide via the clathrin pathway together with claudin-1 (Zwanziger et al., 2012b). As another approach, siRNA against claudin-1 was positively tested in the same model (Hackel et al., 2012, collaboration H. Rittner/Würzburg). In con-clusion, novel tools were developed to improve the delivery of pharmaceutical agents through the perineurial barrier by transient

modulation of claudin-1. To affect claudin-expressing tumor cells, a peptide ligand of the second extracellular loop of claudins 3 and 4, the Clostridium perfringens enterotoxin, demonstrated antitu-mor activity in vitro and in vivo (Walther et al., 2012, collaboration MDC Berlin). In addition, we provided evidence that caprate, a known drug delivery enhancer, reduced the trans-interactions of claudin-5, indicating TJ-related efficacy (Del Vecchio et al., 2012).

Methodological goals:Powerful protein-protein binding assays were advanced or evolved. We discovered and quantified direct heterophilic as-sociations between different claudins and different TAMPs as well as between claudins and TAMPs, ZO proteins, etc. in fluorescence resonance energy transfer procedures (Rückert et al., 2012), trans-interaction assays, fluorescence recovery after photobleaching or TJ diffusibility (Cording et al., 2012; Piontek et al., 2011). Then we developed the first light microscopic approach (superresolution) to allow analyses of TJ networks up to a localization accuracy of

~20 nm and discrimination between single molecules and poly-meric strands in transfected epithelial cells. Quantification of clas-sic claudin networks revealed two populations of elliptic meshes (<100 nm and 300–600 nm; Kaufmann et al., 2012, collaboration C. Cremer/Mainz). These studies are very promising and will be ex-panded to include endothelial cells expressing TJs endogenously. Finally, efforts were made to confirm the in vitro data in vivo as, for instance, in Zebra fish (Zhang et al., 2012, collaboration S. Sey-fried/MDC) or in the development of claudin-12 knock-out mice.

Translation:Several approaches were proven for practical application, result-ing in claudin peptides specifically and transiently opening neu-rological barriers. A European patent (PCT/EP2012/066387) on a

“Peptide agent for enhanced drug delivery for improved periph-eral analgesia” was registered. Here, we defined claudin peptido-mimetics, allowing the delivery of hydrophilic anesthetics to pe-ripheral nerves. The aim is to improve pain alleviation after surgery on extremities (collaboration H. Rittner/Würzburg). Further patent applications were prepared.

Jan Rossa,

Jörg Piontek,

Anna Piontek (Veshnyakova)

72 73

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S G R U P P E N / / / M O L E K U L A R E P H Y S I O L O G I E U N D Z E L L B I O L O G I E

Nadja Käding,

Christian Staat

Olga Breitkreuz-Korff,

Nora Gehne

S E L E C T E D P U B L I C AT I O N S Cording J, Berg J, Käding N, Bellmann C, Tscheik C, Westphal JK, Milatz S, Gunzel D, Wolburg H, Piontek J, Huber O, Blasig IE (2012) Tight junctions: Claudins regulate the interactions between occlu-din, tricellulin and marvelD3, which, inversely, modulate claudin oligomerization. J Cell Sci (electronic publication ahead of print, doi: 10.1242/jcs.114306). Häckel D, Krug SM, Sauer RS, Mousa SA, Böcker A, Pflücke D, Wrede EJ, Kistner K, Hoffmann T, Niedermirtl B, Sommer C, Bloch L, Huber O, Blasig IE, Amasheh S, Reeh PW, Fromm M, Brack A, Rittner HL (2012) Transient opening of the perineurial barrier for analgesic drug delivery. Proc Natl Acad Sci USA 109: E2018-2027. Zwanziger D, Häckel D, Staat C, Böcker A, Brack A, Beyermann M, Rittner H, Blasig IE (2012) A peptidomimetic tight junction modu-lator to improve regional analgesia. Mol Pharm 9: 1785-1794. Blasig IE, Bellmann C, Cording J, Del Vecchio G, Zwanziger D, Huber O, Haseloff RF (2011) Occludin protein family: oxidative stress and reducing conditions. Antiox Redox Signal 15: 1195-1219. Piontek J, Fritzsche S, Cording J, Richter S, Hartwig J, Walter M, Yu D, Turner JR, Gehring C, Rahn HP, Wolburg H, Blasig IE (2011) Elucidating the principles of the molecular organization of hetero-polymeric tight junction strands. Cell Mol Life Sci 68: 3903-3918.

FMP authors Group members

E X T E R N A L F U N D I N G Deutsche Forschungsgemeinschaft, „Alterationen von Occlu-din bei oxidativem Stress und Aufklärung der Funktion von Claudin-12“, FOR 721/2, TP5 (BL 308/9-1), 01.2010 – 06.2013, 126.000 Euro Deutsche Forschungsgemeinschaft, „Modulation der Claudino-ligomerisierung zur Beeinflussung der Blut-Hirnschranke“, BL 308/7-4, 01.2009 – 06.2013, 116.000 Euro (inklusive 1xE13 50%) Deutsche Forschungsgemeinschaft, „Molekulare Organisation von heteropolymeren Tight-Junction-Strängen“, PI 837 / 2-1, with J. Piontek, 07.2009 – 06.2013, 228.000 Euro (inklusive 1xE13) Europäische Kommission, 7. Framework Programme, Project: JUSTBRAIN, „Blood-brain barrier junctions as targets for para-cellular drug delivery to the brain“, 11.2009 – 10.2013, 606.925 Euro B4B Foundation, „Modifying arylsulphatase A with peptides to bypass the blood-brain barrier“, 07.2010 – 12.2013, 80.000 Euro Else-Kröner-Fresenius-Stiftung, „Neue molekulare Therapiean-sätze in der Schmerztherapie durch Öffnung der peripheren Nervenbarriere mittels Tight Junction modulierenden Pepti-den“, 04.2011 – 03.2014, 108.000 Euro Projekt Pasteur Paris, „Definition and validation of surrogate biological markers of neuropathology in the cerebrospinal fluid for mucopolysaccharidosis III”, 01.2011 – 01.2012, 75.000 Euro ProFit Projekt Berlin, IBB, „Blut-Hirnschrankenmodulatoren“, 07.2009 – 12.2011, 274.219 Euro

R E S E A R C H G R O U P S / / / M O L E C U L A R P H Y S I O L O G Y A N D C E L L B I O L O G Y

G R O U P M E M B E R S Dr. Jörg Piontek (group leader) Dr. Rosel Blasig Dr. Giovanna Del Vecchio * Dr. Reiner Haseloff Dr. Lars Winkler * Dr. Denise Zwanziger * Christian Bellmann (doctoral student) * Olga Breitkreutz-Korff (doctoral student) * Victor Manuel Castro Villela (doctoral student) *

Jimmi Cording (doctoral student) Sebastian Dabrowski (doctoral student) Miriam Eichner (doctoral student) * Nora Gehne (doctoral student) * Hans-Christian Helms (doctoral student) * Anja Kublik (doctoral student) * Inga Newie (doctoral student) * Jonas Protze (doctoral student) Jan Rossa (doctoral student) Christian Staat (doctoral student) *

Christian Tscheik (doctoral student) * Barbara Eilemann (technical assistant) * Ramona Günther (technical assistent) * Bianca Hube (technical assistent) * Group members as of 31.12.2012 * Part of reporting period

Fig. 2: Scheme of a peptidomimetic agent taken

out of an extracellular loop of a classic claudin

causing transient opening of the paracellular

cleft in tissue barriers to improve drug delivery.

Example: Peptide C1C2 from murine claudin-1.

C O L L A B O R AT I O N S International EU FP7 collaborative project consortium ‘J U S T B R A I N ’ European consortium ‚Brains4brain’ Anuska AndjelkovicUniversity of Michigan Medical School, Ann ArborUSA Matthew CampbellTrinity College Dublin, Ireland Maria DeliBiological Research Centre, Szeged, Hungary Jean-Michel HeardInstitut Pasteur, Paris, France

Danica StanimirovicInstitute of Biological Sciences, National Research Council, Ottawa, Canada Tetsuya TerasakiTohoku University, Sendai, Japan Jerry Turner, University of Chicago, USA Elga de Vries, Neuroscience Campus, Am-sterdam, Netherlands National DFG Forschergruppe 721 Tight Junctions Cristoph CremerUniversität Heidelberg

Klaus GastUniversität Potsdam Otmar HuberUniversitätsklinikum Jena Bernd NürnbergUniversitätsklinikum Tübingen Heike RittnerUniversitätsklinikum Würzburg Matthias SchroeterMax-Planck-Institute for Human Cognitive and Brain Sciences, Leipzig Wolfgang WaltherMax-Delbrück-Center for Molecular Medicine, Berlin Hartwig WolburgEberhard-Karls-Universität Tübingen

Fig. 1: Interaction possibilities between the scaf-

folding protein ZO-1, protein binding domains

indicated, and other proteins forming the tight

junction assembly.

74 75

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2R E S E A R C H G R O U P S / / / M O L E C U L A R P H Y S I O L O G Y A N D C E L L B I O L O G Y F O R S C H U N G S G R U P P E N / / / M O L E K U L A R E P H Y S I O L O G I E U N D Z E L L B I O L O G I E

M O L E C U L A R P H A R M A C O L O G Y A N D C E L L B I O L O G Y

M O L E K U L A R E P H A R M A K O L O G I E U N D Z E L L B I O L O G I E

G R O U P L E A D E RP R O F. D R . V O L K E R H A U C K E

B I O G R A P H Y

1989 – 1994 Studies in Biochemistry, Freie Universität Berlin and the University of Basel (Biozentrum)

1994 – 1997 PhD student, Department of Biochemistry (Prof. G. Schatz), Biozentrum, University of Basel

1997 PhD (summa cum laude)

1997 – 1999 Postdoctoral fellow, Yale University School of Medicine and HHMI (Prof. P. De Camilli), New Haven

2000 – 2003 Independent group leader, Center for Biochemistry and Molecular Cell Biology, University of Göttingen

2003 – 2005 Professor of Membrane Biochemistry, Freie Universität Berlin

2005 – 2008 Full Professor and Chair (W3), Department of Membrane Biochemistry, Freie Universität Berlin

since 2007 Member of Neurocure Cluster of Excellence

2008 – 2011 Full Professor and Chair (W3), Membrane Biochemistry, Freie Universität and Charité – Universitäts- medizin, Berlin

2008 – 2010 Speaker, Collaborative Research Center (SFB) 449

2011 – 2012 Speaker, Collaborative Research Center (SFB) 958

since 2012 Director of the FMP, Head of the Department of Molecular Pharma-cology & Cell Biology at the FMP, Full Professor of Molecular Pharmacology (S-W3), Institute of Pharmacy, Freie Universität Berlin; co-optated to the Charité – Universitätsmedizin, Berlin

S U M M A R Y

Membrane dynamics within the endocytic and endosomal system play a crucial role in cell physiology and membrane homeostasis, cell signaling and development, the func-tioning of the nervous system, and diseases such as cancer. Research within the depart-ment focuses on the molecular mechanisms of endocytic and endosomal membrane traffic using a wide arsenal of techniques that range from in vitro approaches to in vivo studies. We are particularly interested in the exo-endocytic cycling of synaptic vesicles at neuronal synapses and its role in brain function and disease. An important aspect of these studies is to determine how events at the molecular and subcellular levels trans-late into the functions of individual cells and of cellular networks within the context of an entire organism. To achieve this we are developing molecular tools to dissect and ma-nipulate exo-endocytic cycling or endosomal membrane dynamics, using genetic, bio-chemical, and pharmacological approaches, and by further developing high-resolution imaging techniques. The long-term goal of our work is to unravel the molecular basis of endocytic and endosomal function and dysfunction, thereby opening new avenues for pharmacological interference.

Z U S A M M E N FA S S U N G

Dynamische Membranprozesse des endozytotischen und endosomalen Systems spielen eine entscheidende Rolle in der Zellphysiologie und Membranhomöostase, bei der Sig-nalübertragung zwischen Zellen und der Zellentwicklung, für die Aktivitäten des Nerven-systems und bei Krankheiten wie Krebs. Der Forschungsschwerpunkt unserer Abteilung liegt auf den molekularen Mechanismen des endozytotischen und endosomalen Memb-rantransports, die wir mit einem breiten Spektrum an Techniken, von in vitro Ansätzen bis zu in vivo-Studien, untersuchen. Besonders interessiert sind wir am exo-endozytotischen Zyklus synaptischer Vesikel an neuronalen Synapsen und dessen Rolle bei der Gehirn-funktion und bei Erkrankungen des Nervensystems. Ein wesentlicher Aspekt dieser Unter-suchungen besteht darin, zu ermitteln, wie Ereignisse auf molekularer und subzellulärer Ebene in Funktionen einzelner Zellen und zellulärer Netzwerke innerhalb eines Gesamt-organismus übersetzt werden. Um dies zu erreichen, entwickeln wir mit Hilfe genetischer, biochemischer und pharmakologischer Ansätze und durch Weiterentwicklung hochauf-lösender bildgebender Verfahren molekulare Werkzeuge zur Analyse und Manipulation des Exo-Endozytose Zyklus synaptischer Vesikel sowie der endosomalen Membrandy-namik. Das langfristige Ziel unserer Arbeit ist es, die molekularen Grundlagen der endo-zytotischen und endosomalen Funktion und Dysfunktion zu enträtseln und dabei neue Wege für pharmakologische Eingriffe zu eröffnen.

D E S C R I P T I O N O F P R O J E C T S

Research within the department is conducted within several sub-groups (led by M. Krauss, T. Maritzen, and J. Schmoranzer) and covers five major areas: (i) the cycling of synaptic vesicle (SV) membranes at neuronal synapses; (ii) the mechanism of endocy-tosis; (iii) the regulation of the endolysosomal system including autophagy by spatiotemporally controlled synthesis and turnover of phosphoinositides (PIs); (iv) the role of the cytoskeleton in endo-somal function, cell signalling, migration, and polarized secretion; and (v) the development and application of super-resolution light microscopy techniques to study the above processes.

Synaptic vesicle exo- and endocytosisNeurotransmission involves the calcium-triggered exocytic re-lease of neurotransmitters from synaptic vesicles (SVs) at presyn-aptic active zones, followed by their endocytic recycling. Using mouse knockout technology, Drosophila mutants (with Stephan J. Sigrist, FU Berlin), and RNA interference in combination with op-tical imaging including optogenetics and electrophysiology, we aim to dissect the pathways and molecular mechanisms of SV re-cycling and regeneration. A key question is how exo- and endocy-tosis are coupled and how membrane homeostasis is maintained.

We have identified endocytic adaptors (e.g. stonins, AP180, CALM) that facilitate endocytic sorting of select SV proteins dur-ing exo-endocytic cycling of SVs. Furthermore, we have begun to unravel molecular links between the fusion machinery at active zones and the endocytic system. The consequences of loss of function of these factors on neurotransmission are currently being dissected at the cellular and organismic levels using genetic and optical-biophysical approaches. These studies bear important im-plications for the understanding of neurological disorders and for neurodegeneration.

Mechanisms of endocytosisEukaryotic cells internalize nutrients, antigens, growth factors, pathogens, ion channels and receptors via endocytosis. We are in-terested in determining the exact function of endocytic adaptors and scaffolds including clathrin, AP-2, intersectins, and bin/rvs/amphiphysin homology (BAR) domain proteins in the spatiotem-poral regulation of clathrin-mediated endocytosis using cell bio-logical, chemical, and genetic techniques. Our lab has identified and characterized the first chemical inhibitors of clathrin function as well as endocytic adaptors for cargo sorting and their interac-tion with cargo proteins and lipids. Recent work has also dealt with the question of how membrane deformation in endocytosis is coupled to dynamin-mediated fission and how dynamin assem-

bly is coupled to endocytic vesicle formation. Other projects aim to unravel factors mediating clathrin-independent endocytosis.

Phosphoinositides within the endolysosomal systemPIs serve as spatiotemporal landmarks within the endolysosomal system and for intracellular membrane traffic in general. We have discovered PI kinases and phosphatases with key roles in en-docytosis and in endolysosomal membrane homoestasis includ-ing autophagy. Genetics, RNA interference, and acute chemical and optogenetic perturbations are used to dissect how specific PIs affect the progression of endocytosis and how PI conver-sion along the endolysosomal pathway is achieved. Furthermore, we use biochemical, lipidomic, and proteomic approaches to identify and characterize effectors and regulators of PI turnover and conversion. We also seek to identify novel pharmacological or chemical inhibitors of select PI metabolizing enzymes. These studies bear implications for disease, as PI metabolizing enzymes, e.g. PI 3-kinases or myotubularin PI phosphatases, are implicated in cancer as well as hereditary disorders such as Charcot Marie Tooth disease.

Role of the cytoskeleton in endosomal function, cell signalling, migration, and polarized secretionRecent work from our lab has identified key connections between the endosomal system and microtubules as well as the actin cyto-skeleton. Reciprocal relationships between the cytoskeleton (e.g. actin, microtubules, septins) and the endosomal system regulate cellular functions ranging from nutrient uptake and signaling to cell motility. By employing cell biological and optical techniques in conjunction with mouse genetics we dissect the role of mem-brane scaffolds, small G proteins, and adaptors in regulating cy-toskeletal dynamics, cell signaling and migration, or polarized secretion.

Super-resolution light microscopyRecent advances have allowed us and others to break the dif-fraction barrier of light microscopy. We have further developed a multi-color STORM/PALM-based super-resolution microscopy platform for the imaging of fixed and live samples, e.g. the exo-/endocytic machineries at presynaptic terminals. Furthermore, we employ sensitive multicolor total internal reflection (TIRF) and multicolor 3D structured illumination (SIM) microscopy to visualize the nanoscale organization of the exo-endocytic and endosomal systems.

Katrin Diesenberg, Michael Krauß, Claudia Gras York Posor, Tanja Maritzen, Seong Joo Koo

76 77

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S G R U P P E N / / / M O L E K U L A R E P H Y S I O L O G I E U N D Z E L L B I O L O G I E

Martin Lehmann, Jan Schmoranzer

S E L E C T E D P U B L I C AT I O N S Maritzen T, Zech T, Schmidt MR, Krause E, Machesky LM, Haucke V (2012) Gadkin negatively regulates cell spreading and motility via sequestration of the actin-nucleating ARP2/3 complex. Proc Natl Acad Sci U S A 109: 10382-10387. von Kleist L, Stahlschmidt W, Bulut H, Gromova K, Puchkov D, Robertson M, MacGregor KA, Tomlin N, Pechstein A, Chau N, Chircop M, Sakoff J, von Kries J, Saenger W, Kräusslich H-G, Shu-pliakov O, Robinson P, McCluskey A, Haucke V (2011) Role of the clathrin terminal domain in regulating coated pit dynamics re-vealed by small molecule inhibition. Cell 146: 471-484. Faelber K, Posor Y, Gao S, Held M, Roske Y, Schulze D, Haucke V, Noe F, Daumke O (2011) Crystal structure of nucleotide-free dyna-min. Nature, 477: 556-560. Haucke V, Neher E, Sigrist SJ (2011) Protein scaffolds in the cou-pling of synaptic exocytosis and endocytosis. Nat Rev Neurosci. 12: 125-136. Koo SY, Markovic S, Puchkov D, Mahrenholz C, Beceren-Braun F, Maritzen T, Dernedde J, Volkmer R, Oschkinat O, Haucke V (2011) SNARE motif-mediated sorting of synaptobrevin by the endocytic adaptors CALM and AP180 at synapses. Proc. Natl. Acad. Sci. USA 108: 13540-13545. FMP authorsGroup members

E X T E R N A L F U N D I N G Deutsche Forschungsgemeinschaft, „Funktionelle Charakter-isierung der Adaptorproteine Stonin 1 und γ-BAR beim intra-zellulären Membrantransport“, HA 2686/ 3-2, with T. Maritzen, 09.2009 – 09.2013, 458.400 Euro Deutsche Forschungsgemeinschaft, SFB 958, A01, „Structural and functional organization of endocytic scaffolds within the periac-tive zone“, with T. Maritzen, 07.2011 – 06.2015, 520.360 Euro Deutsche Forschungsgemeinschaft, SFB 958, A07, „Regulation of SH3 domain-containing scaffolds in synaptic vesicle clustering“, with C. Freund, 07.2011 – 06.2015, 410.400 Euro Deutsche Forschungsgemeinschaft, SFB 958, A11, „Structural and functional analysis of septin scaffolds mediating endosomal membrane trafficking“, to M. Krauss, 07.2011 – 06.2015, 413.888 Euro Deutsche Forschungsgemeinschaft, SFB 958, Z02, „Super-resolu-tion light microscopy to resolve nanoscale molecular struc-tures“, to J. Schmoranzer, 07.2011 – 06.2015, 403.600 Euro Deutsche Forschungsgemeinschaft, „Structured illumination Mi-croscopy System“, INST 130/80-1-FUGG, with J. Schmoranzer, 01.2012 – 12.2012, 450.000 Euro Deutsche Forschungsgemeinschaft, „STORM/PALM Super-Reso-lution Mikroskopie System“, INST 130/792-1-FUGG, with J. Schmoranzer and S.J. Sigrist, 08.2010 – 07.2011, 122.600 Euro Deutsche Forschungsgemeinschaft, SFB 740, C08, „Funktionelle Charakterisierung der Assemblierung und Dynamik PI4-Kinase-basierter Module wahrend der Proteinsortierung an endo-somalen Membranen“, with M. Krauss, 01.2011 – 12.2014, 591.200 Euro

Deutsche Forschungsgemeinschaft, SFB 765, B04, „Multivalente Modulation der Clathrin vermittelten Rezeptorendozytose“, 01.2012 – 12.2015, 414.400 Euro Deutsche Forschungsgemeinschaft, FOR 806, B04, „Functional analysis of cell signaling events following inhibition of clatherin /AP2-mediated endocytosis“, HA 2686/3-2, 02.2010 – 01.2013, 428.500 Euro Deutsche Forschungsgemeinschaft, Excellence Initiative, EXC-257 NeuroCure „Towards a better outcome of central nervous sys-tem disorders“, 01.2009 – 12.2011, 90.000 Euro Schram Foundation, „Role of endocytic adaptors and accessory proteins synaptic vesicle protein sorting and recycling“, T287/18544/2008, 05.2009 – 07.2014, 351.600 Euro European Science Foundation (administered via the Deutsche Forschungsgemeinschaft) „Spatio-temporal organization of the synaptic membrane for synaptic vesicle protein recycling (SYN-APSE)“, Euromembrane-SYNAPSE, HA 2686/6-1, 09.2009 – 08.2012, 333.750 Euro Leibniz-Gemeinschaft, Leibniz Vorhaben im Rahmen des Pakts für Forschung und Innovation, „Regulation of cell motility by mem-brane-associated endosomal adaptors“, SAW 2013-FMP-05, to Tanja Maritzen, 01.2013 – 12.2015, 647.052 Euro European Molecular Biology Organization, EMBO Long-Term Fel-lowship, to Marielle C. Grünig, 02.2012 – 01.2015, 69.970 Euro Alexander von Humboldt Foundation, Andrea L. Marat, 11.2012 – 10.2014, 63.200 Euro

R E S E A R C H G R O U P S / / / M O L E C U L A R P H Y S I O L O G Y A N D C E L L B I O L O G Y

G R O U P M E M B E R S Prof. Dr. Michael Krauß (group leader) * Dr. Jan Schmoranzer (group leader) * Dr. Tanja Maritzen (group leader) * Dr. Marielle Eichhorn-Grünig * Dr. Peter Koch * Dr. Gaga Kochlamazashvili * Dr. Natalia Kononenko * Dr. Seong Joo Koo * Dr. Martin Lehmann * Dr. Marta Maglione (joint postdoc with S. J. Sigrist, FU Berlin) * Dr. Andrea Lynn Marat * Dr. Arndt Pechstein *

Dr. Dmytro Puchkov * Jelena Bacetic (doctoral student) *Katharina Branz (doctoral student) * Gala Claßen (doctoral student) * Katrin Diesenberg (doctoral student) * Fabian Feutlinske (doctoral student) * Niclas Gimber (doctoral student) * Claudia Gras (doctoral student) * Burkhard Jakob (doctoral student) * Natalie Kaempf (doctoral student) * Christina Kath (doctoral student) * André Lampe (doctoral student) * Gregor Lichtner (doctoral student) *

Wen-Ting Lo (doctoral student) * York Posor (doctoral student) * Irene Schütz (doctoral student) * Wiebke Stahlschmidt (doctoral student) * Anela Vukoja (doctoral student) * Uwe Fink (technical assistant/ chemist) * Sabine Hahn (technical assistant) * Maria Mühlbauer (technical assistant) * Susanne Thomsen (technical assistant) * Susanne Wojtke (technical assistant) * Group members as of 31.12.2012Group moved to FMP 09.2012 *

Fig. 1: SD-dSTORM of microtubules and clathrin coated pits: NIH 3T3

cells where fixed and stained for microtubules (Alexa 647, Red) and

clathrin heavy chain (Alexa 700, Green) and imaged with SD-dSTORM.

The wide-field (left) is rendered for comparison. Width of microtubules and

clathrin coated pits demonstrate super-resolution beyond the diffraction

limit of the antibody decorated objects. Sizes and scale are marked.

Fig. 2: Mossy fibre synapse from hippocampal CA3 stratum lucidum, scale

bar 1 ηm. SV, synaptic vesicles; PSD, postsynaptic densities. The presynap-

tic nerve terminal is colored in pink, postsynaptic spines in yellow.

C O L L A B O R AT I O N S International Fabio Benfenati, Italian Institute of Technology, Genova, Italy Michael A. Cousin, University of Edinburgh, UK Paolo Di Fiore, FIRC Institute of Molecular Oncology, Foundation (IFOM) & European Institute of Oncology, Milano, Italy Jean Gruenberg, University of Geneva, Switzerland Ari Helenius, ETH Zürich, Switzerland Emilio Hirsch, University of Torino, Italy Josef T. Kittler, University College London, UK Laura M. Machesky, Beatson Institute for Cancer Research, Glasgow, UK

Adam McCluskey, University of Newcastle, Australia Phillip J. Robinson, Children’s Medical Research Institute (CMRI), Sydney, Australia Takeshi Sakaba, Kyoto University, Japan Oleg Shupliakov, Karolinska Institute, Stockholm, Sweden Dimitrios Stamou, University of Kopenhagen, Denmark

National Oliver Daumke, Max-Delbrück-Center for Molecular Medicine, Berlin Christian Freund, Freie Universität Berlin Rainer Haag, Freie Universität Berlin

Jürgen Klingauf, University of Münster Tobias MoserGeorg-August-Universität, Göttingen Christian Rosenmund, Charité – Universitätsmedizin Berlin Mike Heilemann, Johann-Wolfgang-Goethe-Universität Frankfurt am Main Dietmar Schmitz, Charité – Universitätsmedizin Berlin Stephan J. Sigrist, Freie Universität Berlin Carsten Schultz, EMBL, Heidelberg Markus C. Wahl, Freie Universität Berlin Felix T. Wieland, Ruprecht-Karls-Universität Heidelberg

WF (rendered) SD-dSTORM

78 79

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2R E S E A R C H G R O U P S / / / M O L E C U L A R P H Y S I O L O G Y A N D C E L L B I O L O G Y F O R S C H U N G S G R U P P E N / / / M O L E K U L A R E P H Y S I O L O G I E U N D Z E L L B I O L O G I E

B E H AV I O U R A L N E U R O D Y N A M I C S

V E R H A LT E N S N E U R O D Y N A M I K

G R O U P L E A D E R S D R . TAT I A N A K O R O T K O VA D R . A L E X E Y P O N O M A R E N K O

B I O G R A P H I E S

Tatiana Korotkova

1995 – 2000 Studied biology and physiology at Lomonosov Moscow State University, Russia

2000 – 2003 Ph.D., Heinrich-Heine- University, Düsseldorf (Prof. H.L. Haas and Prof. J.P. Huston)

2003 – 2004 Post-Doc, Heinrich-Heine- University, Düsseldorf (Prof. H.L. Haas)

2004 – 2009 Post-Doc, University Clinic for Neurology, Heidelberg (Prof. H. Monyer)

2009 – 2012 Postdoc at the FMP (Dr. A. Ponomarenko), NeuroCure Fellow

2012 Junior group leader, FMP/NeuroCure

Alexey Ponomarenko

1995 – 2000 Studied biology and physiology at Lomonosov Moscow State University, Russia

2000 – 2003 Ph.D., Heinrich-Heine- University, Düsseldorf (Prof. H.L. Haas and Prof. J.P Huston)

2003 – 2004 Post-Doc, Heinrich-Heine- University, Düsseldorf (Prof. H.L. Haas)

2004 Internship at Rutgers University (Prof. G. Buzsaki), Newark, USA

2004 – 2009 Post-Doc, University Clinic for Neurology, Heidelberg (Prof. H. Monyer)

since 2009 Junior group leader, FMP/NeuroCure

S U M M A R Y

Our aim is to reveal the contribution of individual neuronal membrane conductancies to the excitability and synchronization of neuronal networks in vivo. Brain synchronization regimes are instrumental for cognitive functions such as learning and memory and play a key role in disorders such as epilepsy. We are focusing on the role of voltage-gated channels and GABAA-receptors in the operation of hippocampal networks using genetic mouse models. We also investigate how cortico-subcortical communication is organized through synchronization. A further goal is to establish how molecular metabolic signals sensed by the brain coordinate multiple vital functions, including food intake and sleep. We study the activity and interactions of specific neurons in the hypothalamus across vigilance states and homeostatic challenges using high-density electrophysiological recordings and optogenetics in freely behaving mice. We aim to gain insights into the neural basis and molecular determinants of vital functions and their pathologies, in par-ticular, obesity and sleep.

Z U S A M M E N FA S S U N G

Unser Ziel ist es, den Beitrag, den die Leitfähigkeit einzelner neuronaler Membranen zur Erregbarkeit und Synchronisation neuronaler Netzwerke in vivo leistet, aufzudecken. Für kognitive Funktionen wie Lernen und Gedächtnis ist Gehirnsynchronisation unabding-bar, sie spielt auch eine Schlüsselrolle bei Erkrankungen wie Epilepsie. Wir konzentrieren uns bei unserer Forschung auf den Einfluss spannungsabhängiger Kanäle und GABAA-Rezeptoren auf das Funktionieren von hippokampalen Netzwerken und nutzen dafür genetische Mausmodelle. Außerdem untersuchen wir, wie die kortikale und subkortikale Kommunikation durch Synchronisation organisiert wird. Ein weiteres Ziel ist es, herauszu-finden, wie vom Gehirn wahrgenommene molekulare Stoffwechselsignale lebenswichtige Funktionen wie Essverhalten und Schlaf/Wach-Rhythmus koordinieren. Mit Hilfe paralle-ler elektrophysiologischer Ableitungen und Optogenetik an sich frei bewegenden Mäu-sen untersuchen wir die Aktivität und Interaktionen spezifischer Neurone im Hypotha-lamus, quer über Vigilanzzustände und homöostatische Herausforderungen. Wir wollen auf diese Weise Einblicke in die neuronalen Grundlagen und molekularen Determinan-ten lebenswichtiger Funktionen und ihrer Pathologien, insbesondere bei Fettleibigkeit und Schlafstörungen, gewinnen.

D E S C R I P T I O N O F P R O J E C T S

Contribution of KCNQ channels to the hippocampal synchronization in vivoWe have investigated the role of KCNQ channels in the control of neuronal excitability in vivo in collaboration with Prof. T. J. Jentsch. This project has focused on channel proteins called KCNQ3 and KCNQ5, which permit a passage of potassium ions in neurons. Mutations in KCNQ3 cause neonatal epilepsy. While these chan-nels are expressed in the hippocampus and affect the excitability of neurons in vitro, their functions in vivo were not known. We found that both channels are important for neuronal excitability in behaving mice, fast network oscillations in the hippocampus as well as precise neural representations of space. Both gamma (30 – 120 Hz) and ripple (140 – 200 Hz) network oscillations in the hippocampal CA1 area in vivo were impaired in mice lacking functional KCNQ3 or KCNQ5 channels. The organization of hip-pocampal output in mice with non-functional KCNQ5 channels was further altered, as indicated by less precise spatial firing of pyramidal cells accompanied by their facilitated burst discharge. Moreover, spatial working memory was impaired in KCNQ5 mu-tants. Our results show that KCNQ5 controls excitability of hippo-campal networks and influences cognitive processes. Role of GABAA-receptor mediated inhibition of hippocampal interneurons in fast network synchronization in vivoAnother project was focused on the role of interneurons in collec-tive neuronal activity in the hippocampus. Population synchrony during fast oscillations has been implicated in memory processing, yet molecular and cellular composition of the ~200 Hz (“ripple”) network oscillator in the hippocampus remains unresolved. We have studied ripple oscillations following genetic ablation of the γ2-subunit of GABAA-receptors in parvalbumin-positive (PV) in-terneurons in collaboration with Prof. H. Monyer, DKFZ, Heidel-berg. This genetic model features an absence of a major coupling pathway between PV-cells – their synaptic inhibition. In contrast to controls, PV-Δγ2 mice displayed in vivo virtually no oscillatory events with leading frequencies in the ripple band. Local field po-tential and neuronal activity during remaining ripple-like events in PV-Δγ2 mice were organized at slower frequencies. Optogenetic stimulation of PV neurons entrains ripple oscillations. These re-sults indicate that the generation of ripple oscillations in the CA1 area requires GABAA-receptor inhibition onto PV-cells.

Cortico-subcortical communication through synchronization: fast oscillations in the lateral septal nucleusOscillations gate information transfer between brain regions. In this project we focused on the operation of the lateral septal nu-cleus (LS), which relays information streams from the hippocampus and neocortex to the hypothalamus. We investigated rhythmic aspects of network activity in the LS and its coordination with the network activity in afferent regions. We have found that local field potential and neuronal discharge in LS displayed intermittent episodes (40 – 120 ms) of synchronization at gamma frequencies. These fast oscillations were behavioral state-dependent and high-ly coordinated within LS. Unexpectedly, LS gamma oscillations were coherent with concurrently recorded gamma oscillations in the medial prefrontal cortex but not the hippocampus, the activ-ity of which co-varied with that in LS largely in the theta rhythm band (5 – 10 Hz). LS gamma oscillations displayed experience-dependent dynamics. We also investigated functions of LS using optogenetic control of its main inputs. Neural basis of behavioral multitasking and coordination by specific hypothalamic circuitsWe further study how activity of genetically-defined hypothalamic neurons and circuits governs initiation and coordination of mul-tiple vital functions, including food intake and sleep. In particular, we study the activity and emergent interactions within and be-tween classes of optogenetically-identified neurons (orexin and MCH-positive cells) in the hypothalamus across innate behaviors, vigilance states and homeostatic challenges. Through this ap-proach, we aim to gain insights into the neural basis of adaptive behavior, as well as mechanisms of obesity and sleep disorders.

Tatiana Korotkova,

Franziska Bender

80 81

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S G R U P P E N / / / M O L E K U L A R E P H Y S I O L O G I E U N D Z E L L B I O L O G I E

S E L E C T E D P U B L I C AT I O N S Maier N, Morris G, Schuchmann S, Korotkova T, Ponomarenko AA, Bohm C, Wozny C, Schmitz D (2012) Cannabinoids disrupt hip-pocampal sharp wave-ripples via inhibition of glutamate release. Hippocampus 22: 1350-1362. Korotkova T*, Fuchs EC*, Ponomarenko AA, von Engelhardt J, Monyer H (2010) NMDA receptor ablation on parvalbumin-pos-itive interneurons impairs hippocampal synchrony, spatial repre-sentations, and working memory. Neuron 68: 557-569 Wulff P*, Ponomarenko AA*, Bartos M, Korotkova TM, Fuchs EC, Bahner F, Both M, Tort AB, Kopell NJ, Wisden W, Monyer H (2009) Hippocampal theta rhythm and its coupling with gamma oscilla-tions require fast inhibition onto parvalbumin-positive interneu-rons. Proc Natl Acad Sci 106: 3561-3566 Racz A*, Ponomarenko AA*, Fuchs EC, Monyer H (2009) Augment-ed hippocampal ripple oscillations in mice with reduced fast exci-tation onto parvalbumin-positive cells. J Neurosci 29: 2563-2568 Ponomarenko AA, Li JS, Korotkova TM, Huston JP, Haas HL (2008) Frequency of network synchronization in the hippocampus marks learning. Eur J Neurosci 27: 3035-3042. FMP authors Group members * equal first author

E X T E R N A L F U N D I N G The Human Frontier Science Program, “Neural basis of behav-ioural multitasking and coordination by specific hypothalamic circuits”, 10. 2012 – 09. 2015, 350.000 US $ Deutsche Forschungsgemeinschaft, Cluster of Excellence Neu-roCure: “Towards a better outcome of central nervous sys-tem disorders”, PI position A. Ponomarenko, 10.2009 – 09.2014, 350.000 Euro Deutsche Forschungsgemeinschaft, Cluster of Excellence Neuro-Cure: “Towards a better outcome of central nervous system dis-orders”, Habilitationsgrant für Nachwuchswissen schaftlerinnen (in-cluding PI position), T. Korotkova, 01.2011 – 10.2013, 268.400 Euro

R E S E A R C H G R O U P S / / / M O L E C U L A R P H Y S I O L O G Y A N D C E L L B I O L O G Y

G R O U P M E M B E R S Dr. Alexey Ponomarenko (principal investigator) Dr. Tatiana Korotkova (principal investigator since 2012) Franziska Bender (doctoral student) * Tugba Özdogan (doctoral student) * Group members as of 31.12.2012 * Part of reporting period

Fig. 1: Hippocampal oscillations in freely behav-

ing mice: theta (5–10 Hz), gamma (40–100 Hz)

and ripple (140–200 Hz).

Fig. 2: Spatial firing of pyramidal cells in the hip-

pocampus is changed in mice with non-functional

KCNQ5, a voltage-gated K+ channel.

C O L L A B O R AT I O N S International Antoine AdamantidisMcGill University, Montreal, Canada Denis BurdakovNational Institute for Medical Research, London, UK

National Thomas J. JentschLeibniz-Institut für Molekulare Pharmakologie (FMP), Berlin Hannah MonyerDKFZ, Heidelberg Regine HeilbronnCharité – Universitätsmedizin Berlin Dietmar SchmitzCharité – Universitätsmedizin Berlin

Alexey Ponomarenko,

Tugba Özdogan,

Maria Gorbati

Tugba Özdogan

Ivy Xiaojie Gao,

Tugba Özdogan,

Maria Gorbati

Marta Carus

82 83

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2R E S E A R C H G R O U P S / / / M O L E C U L A R P H Y S I O L O G Y A N D C E L L B I O L O G Y F O R S C H U N G S G R U P P E N / / / M O L E K U L A R E P H Y S I O L O G I E U N D Z E L L B I O L O G I E

M O L E C U L A R N E U R O S C I E N C E A N D B I O P H Y S I C S

M O L E K U L A R E N E U R O W I S S E N -S C H A F T E N U N D B I O P H Y S I K

G R O U P L E A D E R D R . A N D R E W J . R . P L E S T E D

B I O G R A P H Y

1994 – 1998 M.Sci. 1st Class Hons, Physics, Imperial College, University of London, UK 1998 – 2002 Ph.D. Imperial College (Prof. Franks and Prof. Lieb), University of London, UK 2003 – 2005 Post-Doc, Dept. of Pharmacology (Prof. Colquhoun), University College London, UK 2005 – 2008 Visiting Research Fellow, laboratory of Cellular and Molecular Neurophysiology (Dr. Mayer), NICHD, NIH, Bethesda, USA since 2008 Junior group leader, FMP, Berlin since 2008 Member, Cluster of Excellence NeuroCure

S U M M A R Y

Our main research interest is the glutamate receptors of excitatory synapses. These receptors are essential for brain function and play roles in diseases such as epilepsy, and cognitive and neurodegenerative disorders. We are particularly interested in how the composition and properties of receptor complexes determine features of synaptic transmission in the brain in health and disease. To achieve these goals, we manipulate receptors with molecular and chemical biology, including deploying unnatural amino acids in mammalian cells. We examine receptor gating with advanced electrophysiologi-cal methods such as ultra-rapid perfusion and single-channel recording. We complement these approaches with investigations of receptor structure and composition using X-ray crystallography, fluorescence microscopy and biochemistry. Through collaborations, we employ computational approaches to analyze and build novel insights into receptor ac-tivation. A second aspect of our research is to extend these studies to other important components of fast signaling in neurons, such as inhibitory neurotransmitter receptors and voltage-gated ion channels.

Z U S A M M E N FA S S U N G

Der Schwerpunkt unserer Forschung beruht auf Glutamatrezeptoren exzitatorischer Synapsen. Diese Rezeptoren sind essenziell für die Funktion unseres Gehirns und spie-len eine außerordentliche Rolle bei Krankheiten wie Epilepsie sowie kognitiven und neu-rodegenerativen Störungen. Wir konzentrieren uns auf den strukturellen Aufbau dieser Glutamatrezeptoren, insbesondere auf seine Eigenschaften im Komplex aus verschie-denen Untereinheiten, die die Leistungen der synaptischen Transmission im Gehirn bei Krankheit und im gesunden Zustand bestimmen. Dafür manipulieren wir Rezeptoren mit molekularbiologischen und chemischen Methoden, wie z.B. durch den Einbau von unnatürlichen Aminosäuren. In Säugerzellen untersuchen wir das Rezeptor-gating mit-tels neuster elektrophysiologischer Methoden wie ultraschnelle Perfusionsmessung und Einzelkanalmessungen. Ergänzend dazu nutzen wir Methoden wie Röntgenstrukturana-lyse, Fluoreszensmikroskopie und Biochemie, um die Rezeptorstruktur zu untersuchen. In Zusammenarbeit mit anderen Gruppen wenden wir computergestütze Methoden an, um Erkenntnisse über die Rezeptoraktivierung zu analysieren. Ein weiterer Aspekt unse-rer Forschung ist die Ausweitung dieser Studien auf weitere Mitspieler/komponenten der schnellen Signalübertragung in Neuronen, die inhibitorischen Neurostransmitter-Rezep-toren sowie spannungsabhängige Ionenkanäle.

D E S C R I P T I O N O F P R O J E C T S

Coupling between gating and desensitization in glutamate receptorsNeurons in the brain communicate by releasing neurotransmitters, like glutamate, at specialized junctions called synapses. The preci-sion and timescale of this signaling is determined by the kinetic profiles of different glutamate receptors. Fast glutamate receptors (like the AMPA receptor) activate in hundreds of microseconds, but some slow receptors (like the kainate receptor) can be inac-tivated after glutamate binding for tens or hundreds of seconds. Previous work from the lab showed that the subunit organization is similar for AMPA and kainate receptors (Das et al PNAS 2010). We set out to investigate how such a range of kinetics (six orders of magnitude) can be generated by a family of receptors with a common architecture.

The glutamate binding domain is a clamshell with an upper and lower jaw. The lower jaw closes on the glutamate molecule, which is presumed to pull open the integral membrane ion channel to activate the receptor. We used site-directed mutagenesis and patch clamp electrophysiology to show that the lower jaw of the glutamate binding domain (a previously ignored part of the re-ceptor) is responsible for the 100-fold difference in recovery from desensitization between AMPA and kainate receptors (Carbone and Plested, Neuron 2012). We also showed that the same panel of mutants had a corre-lated change in activation properties, suggesting a previously unexpected coupling between gating and desensitization. Single channel recording and kinetic modeling provided evidence that desensitization from the open state curtails AMPA receptor synap-tic currents. This observation suggests that tuning of the lower jaw of the ligand binding domain has optimized AMPA receptors for fast synaptic transmission in the brain. This panel of mutants has a unique spectrum of properties and can be used as a tool to fur-ther investigate several biophysical aspects of glutamate receptor activation including synaptic transmission in vivo and interactions between AMPA receptors and auxiliary subunits.

Miriam Chebli,

Jelena Baranovic

Fig 1:

Left panel

Most GluA2 ligand binding domain mutants have little effect on recovery

(green) but the combination of E713T and Y768R (pink) mutant has very

slow recovery from desensitization.

Right panel

Patch clamp recording of the ET/YR mutant (blue dashed line, wild-type

recovery) showing slow recovery (1.1 s–1).

84 85

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S G R U P P E N / / / M O L E K U L A R E P H Y S I O L O G I E U N D Z E L L B I O L O G I E

S E L E C T E D P U B L I C AT I O N S

Lau AY*†, Salazar H*, Blachowitz L, Ghisi V, Plested AJR*†, Roux B† (2013) A conformational intermediate in glutamate receptor activation. Neuron in press. († corresponding authors)

Miranda P, Contreras J, Plested AJR, Sigworth FJ, Holmgren M, Giraldez T (2013) State-dependent FRET reports calcium- and voltage-dependent gating-ring motions in BK channels. Proc Natl Acad Sci USA 2013 110 (13) 5217-5222.

Carbone AL, Plested AJR (2012) Coupled control of desensitiza-tion and gating by the ligand binding domain of glutamate recep-tors. Neuron 74: 845-857.

Lape R*, Plested AJR*, Moroni M, Colquhoun D, Sivilotti LG (2012) The alpha1K276E startle disease mutation reveals multiple inter-mediate states in the gating of glycine receptors. J Neurosci 32: 1336-1352. FMP authors Group members * equal contribution

E X T E R N A L F U N D I N G

Deutsche Forschungsgemeinschaft, “Trapping the activation mechanism of glutamate receptors”, PL 619/1-1, 09.2010 – 03.2014, 260.000 Euro

Deutsche Forschungsgemeinschaft, Excellence Initiative, EXC-257 NeuroCure “Towards a better outcome of central nervous sys-tem disorders”, 01.2009 – 12.2017, 860.000 Euro

Leibniz-Gemeinschaft, Leibniz Vorhaben im Rahmen des Pakts für Forschung und Innovation, „Illuminating glutamate receptors “, SAW, 01.2012 – 12.2014, 693.000 Euro

Charité, Medical Neurosciences PhD programme, stipend to Vik-toria Klippenstein. 50.000 Euro

Boehringer-Ingelheim Fonds, PhD stipend to Ljudmila Katchan, 2012 – 2014, 100.000 Euro

Human Frontier Science Programme, “Structure and dynamics of desensitization in glutamate receptors”, HFSP Fellowship to Hector Salazar, 07.2011 – 06.2013, 120.000 Euro

European Union, Marie Curie Actions, “Structure and dynamics of desensitization in glutamate receptors”, S-DODGR, Project No: 275987, International Incoming Fellowship to Hector Salazar, 18.000 Euro (declined after 3 months in favour of the HFSP fel-lowship)

R E S E A R C H G R O U P S / / / M O L E C U L A R P H Y S I O L O G Y A N D C E L L B I O L O G Y

G R O U P M E M B E R S

Dr. Anna Carbone Dr. Hector Salazar Dr. Jelena Baranovic * Dr. Vera Martos *(Joint with Prof Dr. Hackenberger) Dr Valentina Ghisi * Viktoria Klippenstein (doctoral student) * Miriam Chebli (doctoral student) * Ljudmila Katchan (doctoral student) * Marcus Wietstruk (technician) Group members as of 31.12.2012 * Part of reporting period

C O L L A B O R AT I O N S

International

Chris AhernUniversity of Iowa, Iowa City, IA, USA Teresa GiraldezUniversidad de La Laguna, Tenerife, Spain Albert LauJohns Hopkins University, Baltimore, MD, USA Benoit RouxUniversity of Chicago, IL, USA Daniel ChoquetInterdisciplinary Institute for Neurosci-ence, Bordeaux, France

National

Oliver DaumkeMax-Delbrück-Center for Molecular Medicine, Berlin

Fig 2:

Upper panel

Wild type AMPA receptors are not inhibited by zinc, but a triple histidine

mutant creates a metal trap (blue star) between glutamate binding do-

mains that freezes the receptor in a partially activated state and inhibits

the glutamate activated current.

Lower panel

Cartoon of four subunits in the receptor (glutamate, orange balls) and

the activation pathway. The structure of the fully active state is currently

unknown.

Hector Salazar,

Mila Katchan

Viktoria Klippenstein,

Anna Carbone

Intermediate states and neurotransmitter receptor activationAlthough it is likely that all signaling proteins traverse metastable intermediates, like stepping stones, en route to full activation, it is only in certain ion channels and enzymes that this phenomenon can be detected directly. We have studied the kinetics of activa-tion intermediates in the glycine receptor (Lape et al 2012 JNS) using single channel recording, but structural correlates of partial-ly activated states are lacking. Because partial agonists are attrac-tive drugs, understanding the structure and chemistry of partially activated states could be hugely useful for rational drug design. The overall structure of glutamate receptors was determined in the resting state by Gouaux’s lab (Sobolevsky et al., Nature 2009), but the nature of the transition to the active, open-channel state remains elusive. We are pursuing several related strategies to measure conformational changes during activation of the recep-tor. One approach is to use reversible crosslinks formed between introduced cysteine amino acids, or metal ion traps constructed from artificially introduced histidine residues. The basic idea is that if a trapping site is built into a dynamic part of the receptor, such as a subunit interface, trapping can occur if the two halves of the site approach sufficiently close. If the ability to trap depends on the functional state, then the insertion site is moving to reach the given state. We use biochemistry to confirm physical crosslinking between subunits. We detect the acute effects of trapping on receptor activity with electrophysiology coupled to fast perfusion. From this information we infer the positions of the subunits with sub-nanometre resolution during the activation cycle.

This approach is particularly powerful when combined with high-resolution structural data from crystallography. Using a crystal structure of an unknown crosslinked state as a template, we have used crosslinks to detect a partially activated state of the GluA2 AMPA receptor that is strikingly different from the resting state (Lau et al., submitted). The intermediate state has a compact arrangement of the four ligand binding sites (Figure 2). We are currently investigating if this arrangement is maintained in other states (e.g. the desensitized state).

86 87

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2R E S E A R C H G R O U P S / / / M O L E C U L A R P H Y S I O L O G Y A N D C E L L B I O L O G Y F O R S C H U N G S G R U P P E N / / / M O L E K U L A R E P H Y S I O L O G I E U N D Z E L L B I O L O G I E

P R O T E I N T R A F F I C K I N G

G R O U P L E A D E R P D D R . R A L F S C H Ü L E I N

B I O G R A P H Y

1982 – 1989 Biology studies at the University of Würzburg

1989 Diploma in Biology

1990 – 1993 PhD thesis on toxin transport in E. coli in the laboratory of Werner Goebel (Department of Microbiology, University of Würzburg)

1995 – 1997 Postdoc in the laboratory of Walter Rosenthal (Department of Pharmacology, University of Gießen); work on the vasopressin V2 receptor

since 1997 Group leader at the FMP; work on the trafficking mechanisms of GPCRs

2002 “Habilitation” in Pharmacology and Toxicology (Charité University Medicine Berlin)

S U M M A R Y

G protein-coupled receptors (GPCRs) are the most important drug targets. The receptors must reach their correct subcellular locations, usually the plasma membrane, to function. Transport is enabled by the secretory pathway and starts with a signal sequence-medi-ated insertion of the receptors into the membrane of the endoplasmic reticulum (ER). GPCRs possess one of two types of signal sequences: N-terminal signal peptides which are cleaved off following ER insertion, or signal anchor sequences which form part of the mature protein. The aim of the Protein Trafficking group is to understand the ER insertion of GPCRs, in particular why some GPCRs possess signal peptides whereas others do not. Another aim is to find novel substances that influence the ER insertion of specific GPCRs and compounds that facilitate receptor folding in this compartment.

Z U S A M M E N FA S S U N G

G-Protein-gekoppelte Rezeptoren (GPCRs) sind die wichtigsten Zielmoleküle für Arznei-mittel. Um korrekt zu funktionieren, müssen die Rezeptoren ihre exakte subzelluläre Posi-tion, normalerweise in der Plasmamembran, einnehmen. Dieser Transport wird über den sekretorischen Weg ermöglicht und startet mit einem Signalsequenz-vermittelten Einbau des Rezeptors in die Membran des endoplasmatischen Retikulums (ER). GPCR’s verfügen über zwei Arten von Signalsequenzen: N-terminale Signalpeptide, die nach dem Einfä-deln in die ER-Membran abgespalten werden, und Signalankersequenzen, die Teil des reifen Proteins sind. Ziel der Protein-Trafficking-Gruppe ist es, den Einbau der GPCR’s in die ER-Membran zu verstehen, insbesondere, warum einige GPCRs Signalpeptide haben, andere nicht. Ein weiteres Ziel ist es, neue Wirkstoffe zu finden, die den Einbau spezifi-scher GPCR’s in die ER-Membran beeinflussen und Substanzen, die die Rezeptorfaltung in diesem Kompartiment erleichtern.

D E S C R I P T I O N O F P R O J E C T S

Functional significance of cleavable signal peptides of GPCRs The ER insertion of GPCRs and other integral membrane proteins is mediated by signal sequences (signal peptides or signal an-chor sequences) and the protein-conducting Sec61 channel. In the case of the corticotropin-releasing factor receptor type 2a (CRF2(a)R), we found a third type of signal sequence, an uncleaved N-terminal “pseudo signal peptide” (PSP). We have assessed the functional significance of this novel GPCR domain and could show that its presence prevents receptor oligomerization (1) and cou-pling to Gi. The CRF2(a)R is consequently expressed as a monomer and is only able to couple to Gs. The homologous CRF1R with its cleaved signal peptide, in contrast, forms dimers and is able to couple to both Gs and Gi. These properties could be transferred in signal peptide exchange experiments. The PSP thus represents the first example of a signal sequence influencing receptor oligo-merization and signal transduction processes. These results are summarized schematically in Fig. 1 (p. 88). We are now analyzing whether the pseudo signal peptide can be used to influence the oligomerization state of other GPCRs. This would be a powerful tool to study the functional significance of GPCR oligomerization. In the period reported, we also studied the signal peptide of the protease-activated receptor 1 (PAR1) and could show that the receptor possesses a conventional and cleaved signal peptide. Surprisingly, however, the sequence encoding the signal peptide was found to stabilize the receptor’s mRNA (2). Since the receptor is expressed mainly in platelets, this sequence may guarantee an increased half life of its mRNA in this nucleus-free environment. Substances influencing ER insertion of GPCRs (cooperation: Peptide Chemistry group) Signal peptides do not have sequence homologies and may thus represent good novel drug targets. The idea is to find substances targeting specific signal peptides and consequently preventing synthesis of specific proteins. A substance pointing in this direc-tion is the cyclodepsipeptide cotransin. It was shown to inhibit the biosynthesis of a small subset of proteins in a signal peptide dis-criminatory manner by preventing stable insertion of the nascent chains into the Sec61 channel at the ER membrane (5 proteins; Garrison et al., Nature 436, 285f, 2005). Cotransin is thus a selec-tive, but not a specific, inhibitor of protein synthesis targeting signal peptides. We have analyzed the cotransin sensitivity of vari-ous GPCRs and have shown that the human endothelin B receptor (ETBR) is cotransin sensitive, too (3). Although new results from the

group show that cotransin is less selective than previously thought, its derivatization may still lead to novel biosynthesis inhibitors for specific proteins. On the other hand, finding complete blockers of the Sec61 channel functioning independent of signal peptides may result in powerful research tools or novel anti-tumour drugs. To this end, we currently analyze a lot of cotransin derivatives for novel properties. Analysis of the mechanism of pharmacological chaperones using fusions with the photoconvertible monomeric Kikume green red (mKikGR) protein (cooperation: Cellular Imaging group)Small molecules called ‘pharmacological chaperones’ may fa-cilitate correct folding and transport of disease-causing mutant membrane proteins in the ER membrane. We have analyzed whether pharmacological chaperones act co-translationally, i.e. during receptor synthesis, or post-translationally after receptor synthesis. To this end, we used misfolded and transport-defi-cient mutants of the vasopressin V2 receptor (V2R) fused with the mKikGR protein and the V2R-specific pharmacological chaperone SR121463B. We could show by a novel microscopic assay that SR121463B acts co- or post-translationally depending on the type of mutation (Fig. 2, p. 88)(4). Other results of the groupResults obtained for the structure-function relationships of the thyrotropin receptor (FMP-integrated project, e.g. ref. 5) are sum-marized in the report of the Structural Bioinformatics and Protein Design group.

Wolfgang Klein,

Anita Kinne

88 89

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S G R U P P E N / / / M O L E K U L A R E P H Y S I O L O G I E U N D Z E L L B I O L O G I E

S E L E C T E D P U B L I C AT I O N S Teichmann A, Rutz C, Kreuchwig A, Krause G, Wiesner B, Schü-lein R (2012) The Pseudo signal peptide of the corticotropin-re-leasing factor receptor type 2A prevents receptor oligomerization. J Biol Chem 287: 27265-27274.

Zampatis DE, Rutz C, Furkert J, Schmidt A, Wüstenhagen D, Ku-bick S, Tsopanoglou NE, Schülein R (2012) The protease-activated receptor 1 possesses a functional and cleavable signal peptide which is necessary for receptor expression. FEBS Lett 586: 2351-2359.

Westendorf C, Schmidt A, Coin I, Furkert J, Ridelis I, Zampatis D, Rutz C, Wiesner B, Rosenthal W, Beyermann M, Schülein R (2011) Inhibition of biosynthesis of human endothelin B receptor by the cyclodepsipeptide cotransin. J Biol Chem 286: 35588-35600.

Ridelis I, Schmidt A, Teichmann A, Furkert J, Wiesner B, Schülein R (2012) Use of Kikume green-red fusions to study the influence of pharmacological chaperones on trafficking of G protein-coupled receptors. FEBS Lett 586: 784-791. Kleinau G, Hoyer I, Kreuchwig A, Haas A K, Rutz C, Furkert J, Worth C L, Krause G, Schülein R (2011) From molecular details of the interplay between transmembrane helices of the thyrotro-pin receptor to general aspects of signal transduction in family a G-protein-coupled receptors (GPCRs). J Biol Chem 286: 25859-25871.

FMP authors Group members

R E S E A R C H G R O U P S / / / M O L E C U L A R P H Y S I O L O G Y A N D C E L L B I O L O G Y

G R O U P M E M B E R S

Dr. Claudia Rutz (lab manager) Dr. Jens Furkert Dr. Antje Schmidt * Wolfgang Klein (doctoral student) Ingrid Ridelis Rivas (doctoral student) * Carolin Westendorf (doctoral student) * Dimitris Zampatis (doctoral student) * Bettina Kahlich (technical assistant)

Group members as of 31.12.2012 * Part of reporting period

Jens Furkert,

Carolin Westendorf

Antje Schmidt,

Ralf Schülein

C O L L A B O R AT I O N S

International

Nikos TsopanoglouUniversity of Patras, Greece Giovanna ValentiUniversity of Bari, Italy National

Ulrike Alexiev Freie Universität Berlin Mathias DregerCaprotec Bioanalytics GmbH, Berlin

Gunnar Kleinau Charité – Universitätsmedizin Berlin Enno KlußmannMax-Delbrück Center for Molecular Medicine, Berlin Richard KroczekRobert-Koch-Institut, Berlin Walter Rosenthal Max-Delbrück Center for Molecular Medicine, Berlin Klaus WeißhartCarl Zeiss MicroImaging GmbH, Jena

Fig. 1: Functional significance of the signal peptides of CRF receptors.

The CRF1R (grey) possesses a conventional cleaved signal peptide and

forms oligomers. It couples to Gs and Gi. The CRF2(a)R (black) possesses

an uncleaved PSP, is expressed as a monomer, and couples only to Gs.

These properties could be transferred in signal peptide swap experiments

(constructs SP2-CRF1R and SP1-CRF2(a)R) (1).

Fig. 2: Substance SR121463B acts co- or post-translationally depending on

the type of receptor mutation. HEK 293 cells were stably transfected with

the folding and transport-defective mutant V2R constructs S167T.mKikGR

and R337X.mKikGR. Green fluorescence of the receptors was completely

photoconverted to red by UV irradiation, leading to a resulting red popula-

tion (already synthesized receptors) and an arising green population (newly

synthesized receptors). After treatment with SR121463B (+) or vehicle (-),

the question of which receptor population could be rescued was analyzed

microscopically. In the case of mutant S167T.mKikGR, the green but

not the red population was rescued, indicating that SR121463B acts only

co-translationally. In the case of mutant R337X.KikGR, however, both recep-

tor populations were rescued, indicating that SR121463B is also able to

act post-translationally here. Arrow: rescued red constructs at the plasma

membrane. Scale bar: 10 µm.

Ingrid Ridelis-Rivas

E X T E R N A L F U N D I N G Deutsche Forschungsgemeinschaft, „Ableitung von Struktur-/Funktionsbeziehungen spezifischer Hemmstoffe der Biosynthe-se G-Protein-gekoppelter Rezeptoren“, SCHU 1116/2-1, 01.2011 – 12.2013, 235.000 Euro

Caprotec Bioanalytics GmbH Berlin

90 91

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2R E S E A R C H G R O U P S / / / M O L E C U L A R P H Y S I O L O G Y A N D C E L L B I O L O G Y F O R S C H U N G S G R U P P E N / / / M O L E K U L A R E P H Y S I O L O G I E U N D Z E L L B I O L O G I E

C E L L U L A R I M A G I N G / E L E C T R O N M I C R O S C O P Y

Z E L L U L Ä R E B I L D G E B U N G / E L E K T R O N E N M I K R O S K O P I E

G R O U P L E A D E R S D R . B U R K H A R D W I E S N E R L A S E R S C A N N I N G M I C R O S C O P Y

D R . D O R O T H E A L O R E N ZE L E C T R O N M I C R O S C O P Y

S U M M A R Y

Since 2006, our group has functioned as a core facility. It is split into two sub-units to pro-vide better technical support: “laser scanning microscopy” and “electron microscopy”.We are open to collaborations with all research groups of the FMP. We complete sub-projects independently and this leads to joint publications with the research groups. The laser scanning microscopy core facilityThe light microscopy facility supports all research groups of the FMP with technology and expertise to study biological samples including living cells, fixed cells, tissue, and solutions. Our central role is to establish single cell techniques and apply diverse microscopic meth-ods to describe cellular signal transduction. Microscopic methods such as FRET (Fluores-cence Resonance Energy Transfer), FRAP (Fluorescence Recovery After Photobleaching) or FCS (Fluorescence Correlation Spectroscopy) are well-established in the center. Addi-tionally, we examine the use of a range of caging compounds in connection with intracel-lular Ca2+ measurements that employ UV- and/or IR-irradiation for the process of uncaging. Furthermore, we are developing opportunities for data analysis by developing novel al-gorithms for biophysical issues (e.g. we create macros for the analysis). We also test and create protocols for the use of new dyes, such as the photoconversion of Kaede. The electron microscopy core facilityThe EM facility of the FMP assists interested groups in visualising cellular architecture and in localising individual proteins at a high resolution. In addition, we support high-resolution imaging of 2D protein crystals and fibril structures. The lab provides standard and advanced specimen preparation techniques. Recently, we have acquired a FEI Tecnai G2 FEG 200kV electron microscope with cryo-equipment and are currently establishing cryo-methods for protein and cell imaging. We established also electron tomography on resin embedded samples.

Z U S A M M E N FA S S U N G

Seit 2006 fungiert unsere Gruppe als Technologieplattform des FMP und ist zur Bereitstellung einer besseren technischen Unter-stützung in die beiden Service-Gruppen „Laser-Scanning-Mikros-kopie” und „Elektronenmikroskopie“ unterteilt.Wir bieten allen Forschungsgruppen am FMP unsere Mitarbeit an. Darüber hinaus bearbeiten wir aber auch als unabhängige Arbeits-gruppe eigene Forschungsprojekte, die dann zu gemeinsamen Publikationen mit den Forschungsgruppen führen. Laser-Scanning-MikroskopieDie zentrale Service-Gruppe Lichtmikroskopie unterstützt alle Forschungsgruppen des FMP mit ihrer Technologie und Kompetenz beim Studium biologischer Proben, einschließ-lich lebender Zellen, fixierter Zellen, Geweben und Lösungen. Unsere zentrale Rolle dabei ist es, Einzelzelltechniken zu eta-blieren und verschiedene mikroskopische Methoden für die Darstellung der zellulären Signaltransduktion anzuwenden. Mikroskopische Methoden wie FRET (Fluoreszenz-Resonanz-Energie-Transfer), FRAP (Fluorescence Recovery After Photo-bleaching, Fluoreszenz-Rückgewinnung nach Photobleichung) oder FCS (Fluoreszenz-Korrelations-Spektroskopie) sind bei uns bestens eingeführt. Daneben untersuchen wir im Zusammen-hang mit intrazellulären Ca2+-Messungen den Einsatz einer gan-zen Reihe von caged-Verbindungen (Verbindungen, die durch das Einbringen einer sogenannten Schutzgruppe, Käfig, bio-logisch inaktiv sind), bei denen für das Uncaging (Abspaltung der Schutzgruppe) UV- und/oder IR-Bestrahlung eingesetzt wird. Außerdem entwickeln wir neue Möglichkeiten der Datenanalyse, indem wir neuartige Algorithmen für biophysikalische Themen ent-wickeln (z.B. Makros für die Analyse) und testen und entwickeln Protokolle für die Anwendung neuer Farbstoffe wie beispielswei-se die Photokonvertierung des Proteins Kaede. ElektronenmikroskopieDie FMP-Service-Gruppe Elektronenmikroskopie unterstützt inte-ressierte Gruppen bei der Visualisierung der Zellarchitektur und Lokalisierung einzelner Proteine in hoher Auflösung. Daneben bie-ten wir auch Unterstützung bei hochauflösenden Aufnahmen von 2D-Proteinkristallen und Fibrillenstrukturen an. Das Labor stellt

Probenpräparationstechniken in Standard- und gehobener Aus-führung bereit. Kürzlich haben wir ein mit Kryotechnik ausgestatte-tes FEI Tecnai G2 FEG 200kV-Elektronenmikroskop erworben und etablieren derzeit Kryomethoden für die Protein- und Zell-Bildge-bung. Ebenfalls eingeführt haben wir die Elektronentomographie für Harz-eingebettete Proben.

D E S C R I P T I O N O F P R O J E C T S

The heptahelical G protein-coupled receptors (GPCRs) are known as important drug targets. Following activation by their ligands, they exert their function via the binding of G proteins and activa-tion of specific signal transduction pathways. Our focus is on the analysis of GPCR oligomerization, whose functional significance is not completely understood. For some GPCRs it is known that oligo-merization modulates receptor transport and/or the dynamics of receptor activation. Most importantly, it is not clear for most of the GPCRs whether they exist exclusively as oligomers or in a certain monomer-dimer ratio (M/D) or whether a given ratio is dynamic. In our group, the oligomerization of GPCRs is analysed by the following biophysical methods: fluorescence-resonance-energy-transfer (FRET), fluorescence-liftime-imaging-microscopy (FLIM) and fluorescence-crosscorrelation-spectroscopy (FCCS). Using these techniques, we are able to determine the ratio of monomers and dimers in the plasma membrane of living cells. In cooperation with the group of Ralf Schülein (Protein-Trafficking, FMP) and Gerd Krause (Structural Bioinformatics, FMP) we have shown that the corticotropin releasing factor receptors type 1 (CRF1R) and type 2a (CRF2(a)R) exhibit differences in their homo-dimerization, despite their very homologous sequence (Teichmann et al. 2012). Whereas 23% of the CRF1R exist as homodimers, the CRF2(a)R shows no re-ceptor interactions, caused by its so-called ‘pseudo signal peptide’. Our aim now is to use this tool to further analyse whether the determined ratio of monomers and dimers is influenced by pro-cesses such as ligand binding or internalization and to gain more information about interaction dynamics. Using single cell and single molecule confocal microscopy we want to determine the functional relevance concerning the amount of GPCR dimers in living cells.

Dorothea Lorenz

92 93

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S G R U P P E N / / / M O L E K U L A R E P H Y S I O L O G I E U N D Z E L L B I O L O G I E

Anke Teichmann,

Burkhard Wiesner

S E L E C T E D P U B L I C AT I O N S Biebermann H, Winkler F, Handke D, Teichmann A, Gerling B, Cameron F, Eichhorst J, Gruters A, Wiesner B, Kühnen P, Krude H, Kleinau G (2012) New pathogenic thyrotropin receptor mutations decipher differentiated activity switching at a conserved helix 6 motif of family A GPCR. J Clin Endocrinol Metab 97: E228-232. Orthmann A, Zeisig R, Suss R, Lorenz D, Lemm M, Fichtner I (2012) Treatment of experimental brain metastasis with MTO-liposomes: impact of fluidity and LRP-targeting on the therapeutic result. Pharm Res 29: 1949-1959. Rossa J, Lorenz D, Ringling M, Veshnyakova A, Piontek J (2012) Overexpression of claudin-5 but not claudin-3 induces formation of trans-interaction-dependent multilamellar bodies. Ann N Y Acad Sc 1257: 59-66. Schaal J, Dekowski B, Wiesner B, Eichhorst J, Marter K, Vargas C, Keller S, Eremina N, Barth A, Baumann A, Eisenhardt D, Hagen V (2012) Coumarin-based octopamine phototriggers and their ef-fects on an insect octopamine receptor. Chembiochem 13: 1458-1464. Teichmann A, Rutz C, Kreuchwig A, Krause G, Wiesner B, Schül-ein R (2012) The Pseudo signal peptide of the corticotropin-releas-ing factor receptor type 2A prevents receptor oligomerization. J Biol Chem 287: 27265-27274. Teichmann A, Schmidt A, Wiesner B, Oksche A, Schülein R (2012) Live cell imaging of G protein-coupled receptors. Methods Mol Biol 897: 139-169.

Schmidt V, Baum K, Lao A, Rateitschak K, Schmitz Y, Teichmann A, Wiesner B, Petersen CM, Nykjaer A, Wolf J, Wolkenhauer O, Willnow TE (2012) Quantitative modelling of amyloidogenic pro-cessing and its influence by SORLA in Alzheimer’s disease. EMBO J 31: 187-200. Christian F, Szaszák M, Friedl S, Drewianka S, Lorenz D, Gon-calves A, Furkert J, Vargas C, Schmieder P, Götz F, Zühlke K, Moutty M, Göttert H, Joshi M, Reif B, Haase H, Morano I, Gross-mann S, Klukovits A, Verli J, Gaspar R, Noack C, Bergmann M, Kass R, Hampel K, Kashin D, Genieser HG, Herberg FW, Willoughby D, Cooper DM, Baillie GS, Houslay MD, von Kries JP, Zimmermann B, Rosenthal W, Klussmann E (2011) Small molecule AKAP-protein kinase A (PKA) interaction disruptors that activate PKA interfere with compartmentalized cAMP signaling in cardiac myocytes. J Biol Chem 286: 9079-9096. Hoppmann C, Schmieder P, Domaing P, Vogelreiter G, Eich-horst J, Wiesner B, Morano I, Ruck-Braun K, Beyermann M (2011) Photocontrol of contracting muscle fibers. Angew Chem Int Ed 50: 7699-7702. Westendorf C, Schmidt A, Coin I, Furkert J, Ridelis I, Zampatis D, Rutz C, Wiesner B, Rosenthal W, Beyermann M, Schülein R (2011) Inhibition of biosynthesis of human endothelin B receptor by the cyclodepsipeptide cotransin. J Biol Chem 286: 35588-35600. FMP authors Group members

R E S E A R C H G R O U P S / / / M O L E C U L A R P H Y S I O L O G Y A N D C E L L B I O L O G Y

G R O U P M E M B E R S

Laser Scanning Microscopy

Anke Teichmann (doctoral student) Jenny Eichhorst (technical assistant)

Electron Microscopy

Dr. Dorothea Lorenz (group leader)Svea Hohensee (research assistant) Martina Ringling (technical assistant) Group members as of 31.12.2012

Fig. 1: Representative auto- and crosscorrelation curves of

the CRF1R derived measurements in the basal membrane

of living HEK293 cells. The receptors were C terminally

fused to GFP and to mCherry, respectively. Transiently

transfected HEK293 cells are represented by the mCherry-

tagged CRF1R in the upper right panel.

C O L L A B O R AT I O N S International

Roger A. JohnsonStony Brook University, New York, USA Peter PohlJohannes Kepler University, Linz, Austria Eduard StefanUniversity of Innsbruck, Innsbruck, Austria Grazzia TammaUniversity of Bari, Bari, Italy Ina UlrichDanube Private University, Krems-Stein, Austria Thomas WaltherHull University, Hull, UK National

Dorothea EisenhardtFreie Universität Berlin Martina FröhlichHumboldt-Universität zu Berlin Katarina JewgenowLeibniz Institute for Zoo and Wildlife research, Berlin Karin MüllerLeibniz Institute for Zoo and Wildlife Research, Berlin

Gunnar KleinauCharité – Universitätsmedizin Berlin Edda KlippHumboldt-Universität zu Berlin Jörg RademannUniversity of Leipzig Alexander WenigCharité – Universitätsmedizin Berlin

Max-Delbrück Center for Molecular Medicine, Berlin: Oliver Daumke Iduna Fichtner Enno Klussmann Bettina Purfürst Salim Seyfried Anje Sporbert Reiner Zeisig

Leibniz-Institut für Molekulare Pharmakologie (FMP): Michael Beyermann Ingolf Blasig Margitta Dathe Gerd Krause Ronald Kühne Jörg Piontek Ralf Schülein

Jenny Eichhorst

Martina Ringling

R E S E A R C H H I G H L I G H T S / / / N M R F Ü R G A N Z E U R O PA

94 95

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2

Chemical Systems BiologyChemische Systembiologie

GROUP LEADER Dr. Ronald Frank

PAGE 110

Mass SpectrometryMassenspektrometrie

GROUP LEADER Dr. Eberhard Krause

PAGE 114

Screening Unit

GROUP LEADER Dr. Jens Peter von Kries

PAGE 118

Protein ChemistryProteinchemie

GROUP LEADER Prof. Dr. Dirk Schwarzer

PAGE 122

Chemical Biology IIChemische Biologie II

GROUP LEADER Prof. Dr. Christian P. R. Hackenberger

PAGE 98

Peptide ChemistryPeptidchemie

GROUP LEADER Dr. Michael Beyermann

PAGE 102

Peptide-Lipid Interaction / Peptide TransportPeptid-Lipid-Interaktion / Peptidtransport

GROUP LEADER Dr. Margitta Dathe

PAGE 106

CHEMICAL BIOLOGY SECTION

BEREICH CHEMISCHE BIOLOGIE

96 97

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2C H E M I C A L B I O L O G Y S E C T I O N B E R E I C H C H E M I S C H E B I O L O G I E

tion and validation of first hits discovered in the small molecule screening of pharmacological targets. The recently established medicinal chemistry group led by Marc Nazaré (formerly Sano-fi) will strongly support this aim using prior expertise gained in the pharmaceutical industry. Concomitantly, the groups of Dirk Schwarzer (Protein Chemistry, now full professor for biochemistry in Tübingen), and Michael Beyermann (Peptide Chemistry, retired) have left the FMP. With the expected filling of the vacant chemical biology chair (formerly held by Michael Bienert) the restructuring process of the section will soon be completed.

Significant effort has been invested to provide professional core facilities, in particular the internationally recognized Screen-ing Unit led by Jens Peter von Kries, which is devoted to high-throughput screening of small molecule and RNAi libraries. Scien-tific highlights of research within the Screening Unit have been the establishment of drug screens with zebra fish embryos and the re-sulting identification of approved drugs, which rescue heart devel-opment. Furthermore, the group has established genome-wide RNA interference in combination with high-content screening for compounds, which are able to rescue defective regulation of cel-lular water content. The Screening Unit also has served as a central anchoring point for the collaborative activities with the Leibniz research network “Drug Research and Biotechnology”, the Helm-holtz consortium “Drug Research Initiative” and the neighboring Max-Delbrück-Center for Molecular Medicine. In addition, the Screening Unit provides a focal point of the Chemical Biology platform of FMP, which itself is the central node of the European initiative EU-OPENSCREEN of the ESFRI roadmap, coordinated at the FMP by Ronald Frank. In April 2013, EU-OPENSCREEN was officially included into the German Roadmap for Large Research Infrastructures, which also documents the willingness of the Ger-man Ministry of Science to financially support the future European infrastructure and upgrade of the Berlin site. Chemical Biology Unit and Screening Unit furthermore will become a joint core facil-ity of the Berlin Institute of Health (BIH).

Overall, the Chemical Biology Section has contributed impor-tant modern chemical techniques and expertise, thus providing privileged access to pharmaceutically active substances through the synthesis, identification/screening, and optimization of new (small) molecule compounds for studying under-explored pro-tein targets. These assets ideally support the core mission of the institute to investigate at the molecular level novel principles for the pharmacological intervention with biological processes that ultimately will lead to new avenues in the treatment of diseases.

und neue Ansätze für den gezielten Wirkstofftransport zu entwi-ckeln. Ein wichtiges Ziel der Chemischen Biologie am FMP ist die chemische Optimierung und Validierung bioaktiver Substanzen (sogenannte „Hits“), welche durch das automatisierte Screening pharmakologischer Ziele mit kleinen Molekülen identifiziert werden. Eine neu geschaffene medizinalchemische Arbeitsgruppe unter Marc Nazaré (vormals Sanofi-Aventis) wird diese Aktivitäten durch seine in der pharmazeutischen Industrie gewonnene Expertise substantiell verstärken. Im Berichtszeitraum schieden die Gruppen-leiter Dirk Schwarzer (Proteinchemie, W3-Professur für Biochemie in Tübingen) und Michael Beyermann (Peptidchemie, Ruhestand) aus. In Kürze kann der Restrukturierungsprozess mit der Besetzung der vakanten Professur für Chemische Biologie (ehemals Michael Bienert) erfolgreich abgeschlossen werden.In den vergangenen Jahren wurden ferner erhebliche Anstrengun-gen zur Optimierung der zentralen Einrichtungen (Core facilities) unternommen. Dazu zählt insbesondere die international anerkann-te „Screening Unit” von Jens Peter von Kries, die sich dem Hoch-durchsatz-Screening von kleinen Molekülen und RNAi-Bibliotheken widmet. Ein außerordentlicher wissenschaftlicher Erfolg konnte bei der Wirkstoffsuche mit Zebrafisch-Embryonen erzielt werden: Diese hat zur Identifizierung von bereits in anderem Zusammen-hang als Medikament zugelassenen Substanzen geführt, die in der Lage sind, eine Fehlentwicklung des Herzens bei gendefizienten Fischen aufzuheben. Ein weiteres Beispiel ist die Kombination von RNA-Interferenz über das gesamte Genom und Wirkstoffsuchen an lebenden Zellen mittels automatisierter Mikroskopie (High-Content Screening). So konnten Substanzen gefunden werden, die eine Fehlregulation des zellulären Wassergehaltes beheben können und somit bei speziellen Formen der Diabetes helfen könnten. Die Screening Unit ist zentrale Drehscheibe für Kooperationen mit dem Leibniz Forschungsnetzwerk „Wirkstoffforschung und Biotechno-logie”, dem Helmholtz-Konsortium „Wirkstoffforschungsinitiative” und dem Max-Delbrück-Zentrum für Molekulare Medizin. Innerhalb des Instituts ist sie Mittelpunkt der „Chemical Biology Unit“, die wiederum im Zentrum der europäischen Forschungsinfrastruktur-initiative EU-OPENSCREEN der ESFRI Roadmap steht. Im April 2013 wurde die von Ronald Frank koordinierte Initiative in die deut-sche Roadmap für große Forschungsinfrastrukturen aufgenommen. Damit bringt das Bundesministerium für Bildung und Forschung seine Bereitschaft zum Ausdruck, EU-OPENSCREEN finanziell zu unterstützen und ihre Berliner Einrichtung konsequent auszubauen. Die „Screening Unit“ und die „Chemical Biology Unit“ werden Teil der gemeinschaftlich organisierten Core Facility des neuen Berliner Instituts für Gesundheitsforschung (BIG).Insgesamt hat der Bereich mit innovativen chemischen Techniken und Kompetenzen zu neuen Forschungsergebnissen beigetragen. Den Wissenschaftlern am Institut ermöglichen die Kollegen der Chemischen Biologie mittels Synthese, Identifizierung und Opti-mierung neuer (kleiner) Wirkstoffkandidaten privilegierten Zugriff auf pharmazeutisch aktive Substanzen, um diese zur Erforschung unbekannter Proteinfunktionen zu nutzen. Diese „state of the art“-Forschungsexpertise unterstützt in idealer Weise die zentrale Mission des Instituts, mit biologischen und chemischen Verfahren auf molekularer Ebene neuartige Wirkprinzipien für pharmakolo-gische Interventionen zu untersuchen, um neue Therapien bei der Behandlung von Krankheiten zu finden.

Research projects in Chemical Biology Section apply innovative synthetic and diagnostic chemical methods to probe the biologi-cal function of cellular target molecules, thereby paving the way towards novel approaches in the pharmaceutical and medicinal sciences. Groups within the Chemical Biology Section work to-wards both the synthesis and identification of novel bioactive molecules of high pharmacological potency, and the develop-ment of new chemical and analytical tools for the functional study of biologically relevant cellular proteins.

During the reporting period, the Chemical Biology Section suc-cessfully advanced its research activities as well as its service facili-ties, which were extended in 2011 into a comprehensive Chemi-cal Biology Unit for the development of qualified small molecule tools serving projects from inside and outside the institute (see page 25).

In Chemical Systems Biology, Ronald Frank and his colleagues work on the cellular protein network regulated by the calcium-sensoring regulatory protein calmodulin. Methodologically, the group further developed and improved the use of their propri-etary chemical microarrays. The Peptide-Lipid Interaction group led by Margitta Dathe has a long-standing expertise in the devel-opment of peptide-modified liposomal carriers and their transfer into clinical applications. In cooperation with the Clinic for Der-matology of the Westfälische Wilhelms-Universität Münster, an enzyme substitution therapy for the rare skin disorder Transglu-taminase 1-deficient autosomal recessive Congenital Ichthyosis was developed, which was granted orphan designation by the European Commission in June 2013. Eberhard Krause, head of the mass spectrometry group continued high resolution proteomic studies, with a particular focus on cell signaling and post-transla-tional modifications of proteins. The expertise of this group has a pivotal role for research at other FMP sections. Furthermore, the research activities of the Chemical Biology Section are intimately interconnected with most groups in the other sections, particularly the Structural Bioinformatics group led by Gerd Krause and the Drug Design group led by Ronald Kühne with its computational chemistry approaches (both in the Structural Biology Section).

During the reporting period the section has also continued its restructuring and has further strengthened its expertise in the areas of Chemical Biology and Medicinal Chemistry. In Decem-ber 2012, Christian Hackenberger was appointed as a Leibniz-Humboldt professor for Chemical Biology and also became acting head of the Chemical Biology Section, as the successor of Ronald Frank. Research within his department, Chemical Biology II, is aimed at the synthesis of modified biological macromolecules, in particular peptides and proteins, by combining advanced tech-niques of organic synthesis and biochemical and biophysical ap-proaches. These chemical tools contributed to the engineering of new pharmaceutically active biopolymers. Examples included small PEGylated peptides with intracellular activity, the identifica-tion of new targets for medicinal chemistry research, e.g. in the area of neurodegenerative diseases and viral infection, and new approaches for targeted drug delivery. An important aim within the Chemical Biology Unit at the FMP is the chemical optimiza-

Wissenschaftler des Bereichs „Chemische Biologie“ gehen neue Wege in der Pharmakologie und Medizin: Mit innovativen chemi-schen Synthesemethoden untersuchen sie biologische Funktionen von Zielstrukturen (Targets), an die Wirkstoffe binden, und ebnen damit den Weg für pharmakologische Eingriffe und zukünftige me-dizinische Anwendungen. Die Wissenschaftler widmen sich hierbei der Synthese und Identifizierung neuartiger bioaktiver Moleküle und der Entwicklung neuer chemischer und analytischer Werk-zeuge für die biologische Forschung, z.B. zum Studium zellulärer Proteine und ihrer Wechselwirkungen.Im Berichtszeitraum hat der Bereich seine Forschung und das Angebot seiner Serviceeinrichtungen erfolgreich betrieben und erweitert: Die dafür notwendigen interdisziplinär zusammenar-beitenden Wissenschaftler wurden 2011 zur „Chemical Biology Unit“ zusammengefasst. Ihr Ziel ist die Entwicklung kleiner, als molekulare Werkzeuge geeigneter Moleküle. Das umfangreiche Serviceangebot steht wissenschaftlichen Projekten inner- wie au-ßerhalb des Instituts zur Verfügung (Seite 25). In der Arbeitsgruppe „Chemische Systembiologie” beschäftig-ten sich Ronald Frank und Mitarbeiter mit Calcium regulierten Funktionen von Calmodulin und den resultierenden zellulären Signalantworten. Methodologisch entwickelte und optimierte die Gruppe außerdem die Anwendung ihrer (urheberrechtlich geschützten) chemischen Microarrays, die auf kleinster Fläche Tausende Substanzen für die Prüfung von biologischen Wechsel-wirkungen enthalten. Die von Margitta Dathe geleitete Gruppe

„Peptid-Lipid-Interaktionen” befasst sich mit der Entwicklung lipo-somaler Trägersysteme („Carrier“) und entwickelte mit der Haut-klinik der Universität Münster eine Enzym-Substitutionstherapie für die seltene Hauterkrankung „Transglutaminase 1-defiziente autosomal rezessive kongenitale Ichthyose“, für welche die Euro-päische Kommission den Status „Arzneimittel für seltene Leiden“ erteilt hat. Eberhard Krause leitet die Gruppe „Massenspektrome-trie”, die sich – mit speziellem Fokus auf Signaltransduktion und posttranslationalen Proteinmodifikationen – der hochauflösenden Proteomik widmet. Die Kompetenz dieser Gruppe spielt eine zen-trale Rolle für alle Bereiche am FMP. Die Aktivitäten des Bereichs sind zudem mit Gruppen anderer Bereiche eng verbunden; enge Beziehungen bestehen zu den Gruppen „Strukturelle Bioinformatik und Proteindesign” (Gerd Krause) und „Wirkstoff-Design” (Ronald Kühne), die computergestützte chemische Ansätze entwickelt (beide „Strukturbiologie“).Im Berichtszeitraum hat der Bereich seinen Restrukturierungspro-zess fortgesetzt und seine Expertise in der Chemischen Biologie und der Medizinalchemie ausgebaut und gestärkt. Im Dezember 2012 wurde Christian Hackenberger als Leibniz-Humboldt-Professor für Chemische Biologie berufen. Er trat die Nachfolge von Ronald Frank als kommissarischer Leiter des Bereichs „Chemische Biolo-gie“ an. Seine Forschung zielt darauf ab, moderne Techniken der organischen Synthese mit biochemischen und biophysikalischen Ansätzen zu kombinieren, um modifizierte biologische Makromo-leküle, insbesondere Peptide und Proteine, zu synthetisieren. Der-art erzeugte chemische Werkzeuge tragen dazu bei, sowohl neue pharmazeutisch wirksame Biopolymere, wie z.B. kleine intrazellulär aktive PEG-Peptide zu konstruieren, als auch neue Zielstrukturen für die medizinische Chemie, speziell in den Bereichen neurode-generativer Erkrankungen und Virusinfektionen, zu identifizieren

C H E M I C A L B I O L O G Y S E C T I O N B E R E I C H C H E M I S C H E B I O L O G I E

98 99

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2R E S E A R C H G R O U P S / / / C H E M I C A L B I O L O G Y F O R S C H U N G S G R U P P E N / / / C H E M I S C H E B I O L O G I E

C H E M I C A L B I O L O G Y I I

C H E M I S C H E B I O L O G I E I I

G R O U P L E A D E RP R O F. D R . C H R I S T I A N P. R . H A C K E N B E R G E R

B I O G R A P H Y

1996 – 1998 Undergraduate studies and prediploma in Chemistry (Albert-Ludwigs-Universität Freiburg)

1998 – 1999 Graduate studies and M.Sc. in chemistry with Prof. Samuel H. Gellman (University of Wisconsin/Madison, USA)

2000 – 2003 Ph.D. research with Prof. Carsten Bolm (summa cum laude) at the RWTH-Aachen

2003 – 2005 Postdoctoral scholar (DAAD and DFG) work with Prof. Barbara Imperiali (Massachusetts Institute of Technology, USA)

2004 Research stay with Prof. Sheena E. Radford (University of Leeds, UK)

2005 – 2006 Junior group leader as Liebig-Scholar (FCI) at Freie Universität Berlin

2006 – 2011 Emmy-Noether-Group (DFG) leader at Freie Universität Berlin

2011 – 2012 Habilitation and Associate Professor (W2) for Bioorganic Chemistry at Freie Universität Berlin

Since 2008 Speaker of the graduate college “Multivalency in Chemistry and Biochemistry” within the SFB 765; member of the SFB 765

Since 2011 Speaker DFG priority program SPP 1623 „Chemoselective Reactions for the synthesis and application of functional proteins“

Since 2012 Leibniz-Humboldt Professor (W3) for Chemical Biology funded by the Einstein Foundation Berlin (5 year appointment)

S U M M A R Y

In the densely packed world of a cell, many signaling pathways that support healthy func-tioning and are disrupted in disease are controlled by the modification of proteins. The most common of these functionalization events are phosphorylation and glycosylation, but increasingly other so-called post-translational modifications such as acetylation and methylation are being identified as important “toggle switches” in health and disease. Chemical biologists increasingly want to selectively control functionalization of proteins in the cell both to study the biological role of post-translational modifications and to decorate proteins with fluorescent moieties that permit their visualization, or molecular tags that permit their purification or immobilization.The Hackenberger laboratory aims to develop new chemoselective organic transforma-tions to synthesize and modify peptides and proteins of central biological relevance. Our main aim is to identify and apply these reactions, which can be combined with biochemi-cal methods to study biological and functional aspects of protein modifications. This interdisciplinary approach ranges across organic chemistry, biochemistry and biophysics, and we hope to contribute to central questions in proteomics research and other biologi-cal and pharmaceutical applications.

Z U S A M M E N FA S S U N G

In der dicht gepackten Welt einer Zelle werden viele Signalwege, die ihre natür-liche Funktionalität unterstützen und bei Krankheit zerstört sind, durch molekula-re Veränderungen an Proteinen reguliert. Am häufigsten sind an diesen „Funktiona-lisierungen“ Phosphorylierung und Glycosylierung beteiligt; vermehrt werden aber auch andere posttranslationale Modifikationen wie Acetylierung und Methylierung als wichtige „Wechselschalter” zwischen Gesundheit und Krankheit identifiziert. Chemi-sche Biologen möchten zunehmend die Funktionalisierung von Proteinen in der Zelle selektiv kontrollieren, um sowohl die biologische Rolle der posttranslationalen Modi-fikationen zu erforschen als auch Proteine mit fluoreszierenden Gruppen zu verse-hen, die ihre Visualisierung ermöglichen. Des Weiteren sollen spezifische moleku-lare Markierungen („Tags“) ihre Aufreinigung oder ihre Immobilisierung erlauben. Unsere Gruppe beabsichtigt, neue chemoselektive organische Transformationen zu ent-wickeln, um Peptide und Proteine mit zentraler biologischer Relevanz zu synthetisieren und zu modifizieren. Das Hauptaugenmerk liegt dabei darauf, die Reaktionen zu ermit-teln und einzusetzen, die zur Untersuchung der biologischen und funktionalen Aspekte der Proteinmodifikation mit biochemischen Methoden kombiniert werden können. Wir hoffen, dass dieser interdisziplinäre Ansatz, der organische Chemie, Biochemie und Bio-physik umfasst, zur Beantwortung zentraler Fragen in der Proteomik und anderen For-schungsbereichen aus der Biologie und Pharmazie beiträgt.

D E S C R I P T I O N O F P R O J E C T S

A New Chemical tool for Protein Modifications: Bioorthogonal Staudinger-Phosphite and Phosphonite ReactionsIn the course of the investigation of a Lewis-acid catalyzed phos-phorimidate-phosphoramidate rearrangement, we discovered a quantitative hydrolysis of the phosphorimidate intermediates upon the addition of water. Building upon this observation, we used the Staudinger-phosphite reaction to convert azides into phosphoramidates either in solution or on the solid support. In a first biological application of this reaction we developed a che-moselective phosphorylation of proteins which allowed, in combi-nation with unnatural protein translation, a site-specific incorpora-tion of a charged phosphoramidate moiety into a protein (Scheme 1), which is recognized by a phospho-Tyr specific antibody. In subsequent studies, we engineered an unsymmetrical version of the Staudinger-phosphite as well as a Staudinger-phosphonite re-action for the chemical lipidation, biotinylation and glycosylation of proteins as well as polymeric materials.

Finally, we demonstrated that the Staudinger-phosphite is an ef-ficient transformation even in a highly crowded bio-environment, such as E.coli lysate. Consequently, we further employed this reaction for an efficient and metal-free PEGylation of a model azido-phenylalanine containing protein, which delivers a new class of branched oligoethylene glycol scaffolds for the stabilization of biopolymers in lysates and in the cytosol.

Probing the impact of posttranslational modifications on pep-tide and protein aggregation: Semi-Synthesis of the Alzheim-er-relevant Tau ProteinIn this project we are studying the structural consequences of posttranslational modification on model peptide sequences. Building upon recent model studies in which we studied the im-pact of phosphorylation on aggregating coiled-coils or b-sheets, we also study the effect of phosphorylation and glycosylation on intrinsically unstructured proteins. A prime example is the neu-ronal Tau protein, which exists in an unstructured soluble form, a microtubule bound state of unknown structure, or a hyperphos-phorylated aggregated state found in neurofibrillar tangles in Alzheimer’s disease.

Currently, we are investigating the complex relation between phosphorylation and aggregation of Tau by generating otherwise inaccessible homogeneously phosphorylated proteins using pro-tein semi-synthesis. Very recently, we have succeeded in the first semi-synthesis of a homogeneously phosphorylated functional

Tau protein. Furthermore, we extend this study to glycosylated Tau to evaluate whether a perturbed balance between phosphoryla-tion and glycosylation (O-GlcNAc) is associated with Tau aggre-gation. Other studies (with Guy Lippens, CNRS Lille), in which synthetic phosphorylated peptides were subjected to enzymatic transformations, have already identified two new O-GlcNAc glyco-sylation sites in Tau and, furthermore, demonstrated the recipro-cal relationship between phosphorylation and O-GlcNAcylation.

Development of new unnaturally modified glycoproteins In collaboration with Prof. Stephan Hinderlich (Beuth-Hochschule) we recently expanded the repertoire of unnaturally modified gly-coproteins by metabolic oligosaccharide engineering. For this purpose C4-substituted ManNAc-derivatives were synthesized and incorporated into novel C7-modified sialic acids in glycopro-teins in cells and in living zebrafish (Fig. 2, p. 100). These novel biopolymers will be investigated for the development of new diagnostic and therapeutic glycoproteins and potentially applied to the in vivo modification of living animal models.

Site-specific functionalization of proteins for the acquisition of multivalent glycoconjugatesIn a combined effort with Prof. Budisa (TU Berlin) we employ a combination of classical site-directed mutagenesis, genetic code engineering and bioorthogonal reactions to deliver chemically modified proteins with carbohydrates installed at specific residues. These protein conjugates are employed in multivalent binding studies within the collaborative research center 765, which sup-port the use of proteins as structurally defined scaffolds for the presentation of an exact number of multivalent ligands.

Nicole Nischan,

Oliver Reimann,

Kristina Siebertz

Dominik Schumacher,

Andrew Grimes

101

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2

100

F O R S C H U N G S G R U P P E N / / / C H E M I S C H E B I O L O G I E

S E L E C T E D P U B L I C AT I O N S Broncel M, Krause E, Schwarzer D*, Hackenberger CPR* (2012) The Alzheimer’s disease-related tau protein as a new target for chemical protein engineering. Chem Eur J 18: 2488-2492.

Möller H, Böhrsch V, Bentrop J, Bender J, Hinderlich S*, Hacken-berger CPR* (2012) Glycan-specific metabolic oligosaccharide en-gineering of C7-substituted sialic acids. Angew Chem 124: 6088-6092; Angew Chem Int Ed 51: 5986-5990.

Serwa R, Wilkening I, Del Signore G, Mühlberg M, Claussnitzer I, Weise C, Gerrits M, Hackenberger CPR* (2009) Chemoselective Staudinger-phosphite reaction of azides for the phosphorylation of proteins. Angew Chem 121: 8382-8387; Angew Chem Int Ed 48: 8234-8239.

Hackenberger CPR*, Schwarzer D* (2008) Chemoselective liga-tion and modification strategies for peptides and proteins. Angew Chem 120: 10182-10228; Angew Chem Int Ed 47: 10030-10074.

Kleineweischede R, Hackenberger CPR* (2008) Chemoselective peptide cyclization by traceless Staudinger ligation. Angew Chem 120: 6073-6078; Angew Chem Int Ed 47: 5984-5988.

FMP authorsGroup members

E X T E R N A L F U N D I N G Deutsche Forschungsgemeinschaft, Priority Programme SPP 1623 “Chemoselective reactions for the synthesis and application of functional proteins”, funds for the coordination of the priority programme, 2012 – 2015, 268.600 Euro

Deutsche Forschungsgemeinschaft, Priority Programme SPP 1623, “Site-specific functionalization of nanobodies: From labelling to cellular uptake”, jointly with H. Leonhardt (LMU München) and C. Cardoso (TU Darmstadt), 2012 – 2015, 195.650 Euro

Boehringer-Ingelheim Stiftung, “Chemoselective Staudinger-re-actions for the modification of peptides and proteins“,“Plus 3”-Programme, 2010 – 2014, 838.000 Euro

Deutsche Forschungsgemeinschaft, SFB 765 B05, “Synthesis of multivalent ligand binding systems via chemoselective sac-charide- and peptide-ligations” (1st funding period 2008 – 2011),

“Site-specific functionalization of proteins for the acquisition of multivalent glycoconjugtes“ (2nd funding period 2012 – 2015), 2008 – 2015, jointly with N. Budisa (TU Berlin), 379.200 Euro

Deutsche Forschungsgemeinschaft, SFB 765 (1st and 2nd funding period 2008 – 2015), Integriertes Graduiertenkolleg des SFB, 2008 – 2015, 879.400 Euro

Freie Universität Berlin, Ideenwettbewerb “Nanoscale”, “Incorpora-tion of IR-labels by unnatural protein translation for the site-specific detection of conformational changes in membrane proteins”, jointly with M. Gerrits (RiNA GmbH) and J. Heberle (FU Berlin), 2010, 30.000 Euro

Freie Universität Berlin, Ideenwettbewerb Biokommunikation, “Site-specific protein phosphorylation by a chemoselective Phos-phite-Staudinger reaction”, jointly with C. Freund (FMP) and M. Gerrits (RiNA GmbH), 2008, 20.300 Euro

Freie Universität Berlin, Projekt des Innovationsfonds, “Semi-Syn-thesis of phosphorylated Tau proteins for the study of pa-thological relevant aggregation phenomena”, jointly with G. Lippens (Université Lille), 2008, 22.000 Euro

Bundesministerium für Bildung und Forschung (BMBF), BMBF-Gly-cobiotechnologie, “Development of new cellular systems for the acquisition of diagnostic and therapeutic glycoproteins”, jointly with S. Hinderlich (Beuth Fachhochschule Berlin), 2007 – 2010, 165.000 Euro

Deutsche Forschungsgemeinschaft, Emmy Noether Programm, “New synthetic methods for naturally modified peptides and proteins, their structural evaluation and biological function”, 2006 – 2013, 1.115.000 Euro

R E S E A R C H G R O U P S / / / C H E M I C A L B I O L O G Y

G R O U P M E M B E R S Dr. Divya Agrawal *Dr. Vera Martos (co-advised with Dr. Plested) *Dr. Olaia Nieto *Dr. Stefan Reinke * Dr. Ina Wilkening *Lukas Artner (doctoral student) *Jordi Bertran (doctoral student) *Andrew Grimes (doctoral student) *Paul Majkut (doctoral student) *Michaela Mühlberg (doctoral student) *Nicole Nischan (doctoral student) *Oliver Reimann (doctoral student) *Simon Reiske (doctoral student) *Dominik Schumacher (doctoral student) *Kristina Siebertz (doctoral student) *Robert Vallée (doctoral student) *Dagmar Krause (technical assistant) *

Group members as of 31.12.2012* Part of reporting period

Fig 1: Site-specfic chemical phosphorylation

of a protein by the Staudinger-phosphite reac-

tion. (Reference: R. Serwa, I. Wilkening, G. del

Signore, M. Mühlberg, I. Claußnitzer, C. Weise,

M. Gerrits, C. P. R. Hackenberger*, Angew.

Chem. 2009, 121, 8382-8387, Angew. Chem.

Int. Ed. 2009, 47, 8234-8239, Chemoselective

Staudinger-Phosphite Reaction of Azides for the

Phosphorylation of Proteins)

C O L L A B O R AT I O N S International Guy LippensCNRS and Université des Sciences et Technologies de Lille, FranceCaroline Smet-NoccaUniversité des Sciences et Technologies de Lille, FranceJim PaulsonThe Scripps Research Institute, La Jolla, CA, USAAndrew UditOccidental CollegeLos Angeles, CA, USAGerhard ObermeyerUniversität SalzburgRoland BrockRadboud University Nijmegen Medical Centre, The NetherlandsKolio TroevInstitut of Polymers, Bulgarian Academy of Sciences, Sofia, BulgariaMichal PietrusiewiczMaria Curie-Sklodowska University Lublin, Poland

National Nedjliko BudisaTechnische Universität BerlinJens DerneddeCharité – Universitätsmedizin BerlinWerner ReutterCharité – Universitätsmedizin BerlinStephan HinderlichBeuth-Hochschule, BerlinMichael GerritsRiNA GmbHVolker HauckeLeibniz-Institut für Molekulare Pharmakologie (FMP) and Freie Universität BerlinEberhard KrauseLeibniz-Institut für Molekulare Pharmakologie (FMP)Harald SchwalbeJohann-Wolfgang-Goethe-Universität Frankfurt am MainCristina CardosoTechnische Universität Darmstadt Heinrich LeonhardtLudwig-Maximilians-Universität München

Fig 2: Glycan-Specific Metabolic Oligosaccharide

Engineering of C7-Substituted Sialic Acids in liv-

ing zebrafish. (Reference: H. Möller, V. Böhrsch,

J. Bentrop, J. Bender, S. Hinderlich,* C. P. R.

Hackenberger*, Angew. Chem. 2012, 124, 6088-

6092, Angew. Chem. Int. Ed. 2012, 51, 5986-

5990, Glycan-Specific Metabolic Oligosaccharide

Engineering of C7-Substituted Sialic Acids)

Olaia Nieto García,

Lukas Artner

102 103

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2R E S E A R C H G R O U P S / / / C H E M I C A L B I O L O G Y F O R S C H U N G S G R U P P E N / / / C H E M I S C H E B I O L O G I E

P E P T I D E C H E M I S T R Y

P E P T I D C H E M I E

G R O U P L E A D E RD R . M I C H A E L B E Y E R M A N N

B I O G R A P H Y

1971 – 1975 Studied chemistry at the Humboldt University, Berlin

1975 – 1978 Ph.D. (protein semi-synthesis) at the Humboldt University, Berlin

1978 – 1991 Research Associate at the Institute of Drug Research of the Academy of Sciences of the GDR

1988 – 1989 Research Associate at the Department of Chemistry of the University of Massachusetts

1992 – 2012 Head of the Peptide Chemistry group at the FMP

S U M M A R Y

Light is an ideal trigger for the spatiotemporal direction of a biological process in cells because most cells are not affected by light. A main topic of the group has been the design and synthesis of light-controllable, biologically interesting peptides. By incorpo-rating molecular photoswitches we achieved the first amyloid Ab(1-42) analogue show-ing light-controllable toxicity and the first polypeptide ligand of a G protein-coupled receptor whose activity can be switched between agonism and antagonism. Moreover, we have created photo-switchable click amino acids that are incorporated into helical peptide domains to control helicity and, thereby, bioactivity.

Z U S A M M E N FA S S U N G

Licht ist ein idealer Trigger für eine orts-und zeitspezifische Steuerung eines biologischen Prozesses in Zellen, da die meisten Zellen durch Licht nicht beeinflusst werden. Ein zen-trales Thema der Gruppe sind die Planung und Synthese von biologisch interessanten, licht-regulierbaren Peptiden. Durch Einbau molekularer Photoschalter erhielten wir das erste Amyloid Ab(1-42)-Analogon, das eine durch Licht regulierbare Toxizität aufweist, und den ersten Polypeptid-Liganden für einen G-Protein-gekoppelten Rezeptor, dessen Aktivität zwischen Agonismus und Antagonismus wechseln kann. Außerdem haben wir photoschaltbare click-Aminosäuren entwickelt, die in helikale Peptidomänen eingebaut werden, um die Helizität und dadurch die Bioaktivität zu regulieren.

D E S C R I P T I O N O F P R O J E C T S

Photoswitchable biomolecules for in vivo applications are becom-ing increasingly important because they allow the investigation of biological processes with precise spatiotemporal control in a minimally invasive fashion. Recently we have shown that a cellular activity can be directed using a cell-permeable light-switchable peptide ligand bearing an azobenzene-ω-amino acid as a con-formational trigger.

Light-directed formation of molecular bridges in peptides and proteins In the case of proteins their bioactivity is frequently directed by controlling the conformation of certain domains. Photocontrol has been achieved by incorporating two cysteines at an appropriate distance along a helical domain and their bridging with a bifunc-tional (thiol-selective) photoswitch element. This approach cannot work in in vivo systems because of the lack of specificity of the re-action under the conditions of the live cell. We created a concept for the specific formation of a photoswitchable side-chain-to-side-chain bridge in conformational domains of peptides and proteins based on the design of stable photoswitchable click amino acids (PSCaa) bearing an azobenzene unit as side-chain that is elon-gated by a function which can react specifically with thiols – but only after light-activation. We have shown as proof of principle the incorporation of a PSCaa into the polypeptide hormone urocortin 1. The photoswitchable click amino acid is stable during peptide synthesis/purification and allows a subsequent crosslinking to a unique cysteine via light-induced thiol-click reaction under very mild conditions. The intramolecular formation of the photocon-trollable bridge (Fig. 1, p. 105) can proceed even in the presence of other thiols. Provided the PSCaa is incorporated into a protein, the method offers the formation of light-controllable bridges even in live cells. Moreover, with our photoswitchable urocortin ana-logue we have offered a first example for a polypeptide hormone, the biopotency of which can be directed by light.

Light-controlled toxicity of engineered amyloid-b peptidesIn the pathogenesis of Alzheimer Desease (AD) the formation of amyloid plaques derived from the 42-residue long amyloid-b-peptide generated by proteolytic cleavage of the amyloid precur-sor protein (APP) by b- and γ-secretases plays a pivotal role. These plaques consist of fibrillar aggregates. The most neurotoxic forms of Ab are thought to be small oligomers. Such intermediates were found to be responsible for the loss of synaptic function, while

larger Ab fibrils are supposed to be nontoxic. Thus, structural transitions, including the conversion of a-helical conformations into b-pleated sheets, are hypothesized as crucial steps in the pathogenesis of AD.

The investigation of early events in amyloidogenesis or pathogen-esis of AD is hampered by the lack of a conformationally stable, non-toxic form of Ab and the dynamics during the conformational transitions of Ab in solution, which cannot be controlled. Recently, it has been shown for model peptides that trans/cis photoisomer-ization of embedded azobenzene units can be used to direct for-mation and dissociation of fibrils. This observation prompted our approach to incorporate 3-((4’-aminomethyl)phenylazo) benzoic acid into the hydrophobic region of Ab, replacing the VFFA motif that plays a key role in a-to-b transition and fibril formation of Ab (Fig. 2, p. 105). And indeed, the trans photo-Ab formed mature, rod-shaped fibrils, whereas the cis form showed oligomers with a cluster typical of Ab immature protofibrils (TEM by D. Lorenz (FMP)). Most importantly, illumination of mature fibrils of trans photo-Ab induced a complete dissociation of the fibrils and the formation of oligomers. Toxicity was investigated with human neu-roblastoma SH-SY5Y cells (cooperation with G. Multhaup (Freie Universität Berlin)). Treatment of the cells with the mature fibrils revealed no significant reduction of living cells, whilst incubation with cis photo-Ab oligomers resulted in a significant decrease of living cells comparable to that observed with freshly dissolved Ab1-42. Thus, our results show that amyloid fibrils, at least those consisting of photo-Ab, represent a nontoxic state. Upon illumina-tion the Ab fibrils disaggregate, forming soluble, toxic cis photo-Ab oligomers, a result that confirms the often-heard hypothesis that oligomers of Ab can be generated from plaque deposited material and cause cellular death. This is an important finding, especially against the background of vaccination studies in late-disease stages where re-solubilization of aggregated material could lead to increased toxicity and thus severely compromise the generic strategy. In addition, the light-controlled disassem-bly of Ab fibrils into distinct, toxic oligomers offers, for the first time, experimental conditions to start appropriate experiments under controllable conditions from a defined nontoxic specimen that can be converted into toxic ones which may allow for study-ing early events in the pathogenesis of AD with spatiotemporal resolution.

Dagmar Michl,

Nadja Heinrich

104 105

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S G R U P P E N / / / C H E M I S C H E B I O L O G I E

S E L E C T E D P U B L I C AT I O N S Hoppmann C, Barucker C, Lorenz D, Multhaup G, Beyermann M (2012). Light-Controlled Toxicity of Engineered Amyloid beta-Peptides. ChemBiochem 13: 2657-2660.

Kashikar ND, Alvarez L, Seifert R, Gregor I, Jackle O, Beyermann M, Krause E, Kaupp UB (2012). Temporal sampling, resetting, and adaptation orchestrate gradient sensing in sperm. J Cell Biol 198: 1075-1091.

Hoppmann C, Schmieder P, Domaing P, Vogelreiter G, Eichhorst J, Wiesner B, Morano I, Rück-Braun K, Beyermann M (2011) Photo-control of contracting muscle fibers. Angew. Chem. 50, 7699-7702.

Hoppmann C, Schmieder P, Heinrich N, Beyermann M (2011). Pho-toswitchable click amino acids: light control of conformation and bioactivity. Chembiochem 12: 2555-2559.

Piotukh K, Geltinger B, Heinrich N, Gerth F, Beyermann M, Freund C, Schwarzer D (2011). Directed evolution of sortase A mutants with altered substrate selectivity profiles. J Am Chem Soc 133: 17536-17539.

Stefan E, Malleshaiah MK, Breton B, Ear PH, Bachmann V, Beyer-mann M, Bouvier M, Michnick SW (2011). PKA regulatory subunits mediate synergy among conserved G-protein-coupled receptor cascades. Nat Commun 2: 598.

FMP authorsGroup members

E X T E R N A L F U N D I N G Deutsche Forschungsgemeinschaft, “Strukturelle Untersuchun-gen früher Phasen der Fehlfaltung des Proteins beta2-Mi-kroglobulin – ein Beitrag zum Verständnis der Entstehung von Amyloidosen“, NA 226/12-2, with B. Uchanska-Ziegler (Chari-té – Universitätsmedizin Berlin), D. Naumann (Robert-Koch-Institut), 08.2005 – 07.2011, 126.333 Euro

Deutsche Forschungsgemeinschaft, Forschergruppe FG 806 (Teil-projekt 3), „Design , synthesis and functional characteristics of low molecular weight proline-rich motif (PRM) mimetics recog-nized by PRM binding domains”, KU 845/2-2, with R. Kühne, H.-G. Schmalz, 03.2010 – 03.2013, 123.000 Euro

Deutsche Forschungsgemeinschaft, Forschergruppe FG 806 (Teil-projekt 3), „Design , synthesis and functional characteristics of low molecular weight proline-rich motif (PRM) mimetics recog-nized by PRM binding domains”, BE 1434/6-2, with H.-G. Schmalz, R. Kühne, 01.2011 – 12.2013, 15.000 Euro

Deutsche Forschungsgemeinschaft, „Ableitung von Struktur-/Funktionsbeziehungen spezifischer Hemmstoffe der Biosyn-these G-Protein-gekoppelter Rezeptoren“, SCHU 1116/2-1, with R. Schülein, 01/2011 – 06/2014, 235.000 Euro

R E S E A R C H G R O U P S / / / C H E M I C A L B I O L O G Y

G R O U P M E M B E R S Dr. Nadja Heinrich Sabine Abel (doctoral student)Christian Hoppmann (doctoral student) *Angelika Ehrlich (technical assistant) *Annerose Klose (technical assistant) *Dagmar Krause (technical assistant)Dagmar Michl (technical assistant)Bernhard Schmikale (technical assistant)

Group members as of 31.12.2012* Part of reporting period

C O L L A B O R AT I O N S International Jean RivierThe Salk Institute for Biological Studies, La Jolla, USALouis A. CarpinoUniversity of Massachusetts, USA

National Gerd MulthaupFreie Universität BerlinJoachim JankowskiCharité – Universitätsmedizin BerlinKarola Rück-BraunTechnische Universität BerlinHeinz FabianRobert-Koch-Institut, BerlinFrank BernhardJohann Wolfgang Goethe-Universität Frankfurt am Main

Fig. 1: Concept of photoswitchable click amino

acids (PSCaa). PSCaas incorporated into proteins

allow light-directed bridges to be formed for

photocontrol of protein conformation and

activity through a specific light-induced “click”

reaction.

Fig. 2: Photoswitchable amyloid-b-peptide

(photo-Ab): primary structure of native

amyloid-Ab(1-42) and of photo-Ab bearing 3-((4’

aminomethyl) phenylazo) benzoic acid. The cis

form of photo-Ab and the freshly dissolved trans

form are toxic to neuroblastoma cells, while

mature fibrils, formed by the trans form only, are

nontoxic. After light-induced disassembly of the

fibrils, the toxic cis form oligomers are formed.

Bernhard Schmikale

Sabine Abel

106 107

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2R E S E A R C H G R O U P S / / / C H E M I C A L B I O L O G Y F O R S C H U N G S G R U P P E N / / / C H E M I S C H E B I O L O G I E

P E P T I D E - L I P I D I N T E R A C T I O N / P E P T I D E T R A N S P O R T

P E P T I D - L I P I D - I N T E R A K T I O N / P E P T I D T R A N S P O R T

G R O U P L E A D E RD R . M A R G I T TA D AT H E

B I O G R A P H Y

1974 Diploma thesis in physics at the Humboldt-Universität Berlin

1978 Ph.D. at the Academy of Sciences of the German Democratic Republic

1979 – 1992 Research Associate at the Institute of Drug Research

1992 – 1999 Team Leader of the Conformational Analysis Group at the FMP

since 1999 Team Leader of the Peptide Lipid Interaction/Peptide Transport Group at the FMP

S U M M A R Y

Our group is interested in the abilities of peptides to recognize and translocate through membranes and render them permeable. We exploit these properties in the develop-ment of peptides as targeting and uptake-mediating tools for drugs and lipid-based drug carrier systems. Currently, our activities focus on the delivery of drugs through the blood-brain barrier and the efficient cutaneous delivery of bioactive compounds. We are also attempting to elucidate the mode of action of small antimicrobial peptides and optimize their activity for selected applications.

Z U S A M M E N FA S S U N G

Unsere Gruppe interessiert sich für die Fähigkeiten von Peptiden, Membranen zu erken-nen, sie zu überwinden und permeabel zu machen. Wir nutzen diese Eigenschaften zur Entwicklung von Peptiden, die dann als Werkzeuge für den zielgerichteten Transport und die zelluläre Aufnahme von Arzneimitteln und Arzneimittel-Carriern auf Lipidbasis dienen sollen. Derzeit konzentrieren wir unsere Aktivitäten auf die Entwicklung von Trägersys-temen für den Wirkstofftransport über die Blut-Hirn-Schranke und die effiziente Aufnah-me von bioaktiven Substanzen über die Haut. Weiterhin versuchen wir die Wirkungswei-se von kleinen antimikrobiellen Peptiden aufzuklären, um ihre Aktivität für ausgewählte Applikationen zu optimieren.

D E S C R I P T I O N O F P R O J E C T S

Peptide-modified liposomal and micellar carriersThe optimisation of carrier systems in terms of cell selectivity and cellular uptake is crucial for efficient drug delivery. So-called cell-penetrating peptides (CPP), chemically attached or stably adsorbed, mediate efficient uptake of nanoparticles into differ-ent cells. Based on our early studies on a dipalmitoylated short cationic apolipoprotein E sequence (P2A2), we tested the hypoth-esis that selective cellular uptake of peptide-modified particulate systems into brain capillary endothelial cells is determined by an interplay of particle size and surface charge. Generated lipopep-tides of different cationic and anionic amino acid sequences were shown to self-assemble into small micelles at different submicro-molar concentrations and proved to be suitable for the prepa-ration of liposomes of variable size and surface charge density.  Whereas small P2A2 micelles (in contrast to P2A2 liposomes) show selectivity for human brain microvascular endothelial cells (HBMEC) (Fig. 1, p. 109), lysine-rich particles internalize and ac-cumulate non-selectively at the surface of HBMEC and aortic en-dothelial cells, and negatively charged particles locate exclusively on cell surfaces. Contrary to our hypothesis, the peptide sequence proved to be crucial for selective internalization: arginine-rich particles, independent of size and peptide loading, were prefer-entially internalized into HBMEC. Thus, arginine-loaded particles seem to be suitable systems for addressing the blood brain barrier. In cooperation with the University Münster we developed a P2A2-liposomal formulation of recombinantly expressed Transglutamin-ase-1 (TG-1) as the first step of an enzyme replacement therapy to treat TG-1 deficient lamellar ichthyosis, a rare and severe genetic skin disease. Efficient internalization of the liposomes into kerati-nocytes, and considerable improvement of the ichthyosis pheno-type in a topically treated skin-humanized mouse model, suggest this is a promising strategy for restoring epidermal integrity and skin architecture of ichthyosis patients.

Cationic antimicrobial peptidesAntimicrobial peptides (AMPs) have a promising potential as a new class of antibiotics. Knowledge of the principles of action is the prerequisite for optimising their activity and bacterial selectivi-ty (Fig. 2, p. 109). Membrane permeabilisation is the most common mode of action. But, more recently, membrane translocation and interference with intracellular processes like protein synthesis and DNA replication, or demixing of membrane lipids where AMPs interfere with functional proteins and/or disturb cell division, have been suggested as additional mechanisms. Our work focuses on a small arginine (R)- and tryptophan (W)-rich peptide ring with high antibacterial activity. The lack of permea-bilisation of bacterial membranes and of membrane-mimicking lipid bilayers, as well as the peptide-induced disturbance of the metabolism of listeria pathogens and malaria-causing Plasmo-dium species (Cooperation with University Stellenbosch), suggest a special antimicrobial killing mechanism. We synthesized a vari-ety of labelled peptide analogues to be used in Laser Scanning Microscopy, HPLC and Mass Spectrometry to investigate putative translocation mechanisms. Whereas any chemical modification in the W-rich peptide domain distinctly influenced the activity spec-trum and mode of action, substitution of R by conformationally restricted cationic analogues had only minor effects (Cooperation with University Kiev). The results confirm that the most important activity-modulating structural motif is located in the hydrophobic cluster of the peptide and studies with various bacteria and eu-karyotic cells hint at different cellular localizations of the peptides, depending on cell type and peptide concentration.Ongoing collaborations concentrate on proteomic approaches to determine peptide-related changes in protein expression (Ruhr-Universität, Bochum) and real-time analyses of the bacterial fate upon AMP treatment (University of Wisconsin-Madison).Formulations of a cyclic hexapeptide proved efficient in boar semen conservation and were successfully used in artificial in-semination (Cooperation with FLN Schönow). A new potential application of the cyclic peptides results from a surprisingly potent antifungal effect (Cooperation with University Stellenbosch). 

Kathi Scheinpflug,

Heike Nikolenko,

Karl Sydow

108 109

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S G R U P P E N / / / C H E M I S C H E B I O L O G I E

S E L E C T E D P U B L I C AT I O N S Bagheri M, Beyermann M, Dathe M (2012) Mode of action of cat-ionic antimicrobial peptides defines the tethering position and the efficacy of biocidal surfaces. Bioconjugate Chem 23: 66-74.

Arouri A, Kerth A, Dathe M, Blume A (2011) The Binding of an Amphipathic Peptide to Lipid Monolayers at the Air/Water Inter-face Is Modulated by the Lipid Headgroup Structure. Langmuir 27: 2811-2818.

Arouri A, Kiessling V, Tamm L, Dathe M, Blume A (2011) Morpho-logical changes induced by the action of antimicrobial peptides on supported lipid bilayers. J Phys Chem B 115: 158-167.

Bagheri M, Keller S, Dathe M (2011) Interaction of W-substituted analogs of cyclo-RRRWFW with bacterial lipopolysaccharides: the role of the aromatic cluster in antimicrobial activity. Antimicrob Agents Chemother 55: 788-797.

Oehlke J, Ehrlich A, Krause E, Pritz S, Wiesner B, Beyermann M  (2011) Growth factor- and adhesion protein-like components of fetal calf serum can significantly enhance the intracellular delivery of Peptide nucleic acids. Nucleic Acid Ther 21: 285-291.

Oehlke J, Turner Y, Pritz S, Bienert M (2011) Evidence for exten-sive non-endocytotic translocation of peptide nucleic acids across mammalian plasma membranes. Curr Drug Deliv 8: 526-533. FMP authorsGroup members

E X T E R N A L F U N D I N G Deutsche Forschungsgemeinschaft, „Peptide-modified micellar nanocarriers to mediate transport at the blood brain barrier (BBB)”, DA 324/9-1, 06.2010 – 06.2013, 153.000 Euro

Internationales Büro des Bundesministerium für Bildung und Forschung (BMBF) im Projektträger beim Deutschen Zentrum für Luft und Raumfahrt e.V., „Optimising of small cyclic peptides as potential drugs against intracellular human pathogens“ , SUA 09/048, with  M. Beyermann and M. Rautenbach, Stellenbosch University, South Africa, 05.2010 – 04.2013, 18.136 Euro

Alexander v. Humboldt Stiftung, “Synthesis and study of small antimicrobial peptides containing conformationally con-strained arginine analogues”, 2.3-DEU/1136332, with I. Komarov, Shevchenko University, Kiev, Ukraine, 07.2010 – 06.2013, 55.000 Euro

Foundation for Ichthyosis and Related Skin Types, USA, “Liposo-mal Packing of recombinant transglutaminase-1”, with H. Traupe (Universitätsklinikum Münster), 2011 – 2012, 5.000 Euro 

R E S E A R C H G R O U P S / / / C H E M I C A L B I O L O G Y

G R O U P M E M B E R S Dr. Oxana Krylova Dr. Johannes Oehlke *Kathi Scheinpflug (doctoral student)Karl Sydow (doctoral student)Heike Nikolenko (technical assistant)Gabriela Vogelreiter (technical assistant)  Group members as of 31.12.2012* Part of reporting period

Fig. 1: CLSM spectroscopic Studies of the Uptake of Lipopeptide Mi-

celles and Liposomes into Human Brain Microvascular Endothelial Cells

Fluorescence-labeled P2A2 (A) and P2Rn (B) micelles are efficiently

internalized (green). P2A2-tagged liposomes show reduced uptake (C),

but the uptake of P2Rn-modified liposomes is pronounced (D). Trypan

blue in the cell membrane (red) reflects cell viability.

Fig. 2: Modes of Action of Antimicrobial Peptides (AMPs)

Most AMPs kill microorganisms by permeabilising the membrane

which leads to efflux of solvents, loss of membrane potential and

eventual degradation of the cell barrier. More specific antimicrobial

strategies target cytoplasmic processes or lead to demixing and

phase separation of phospholipids with consequences for inte-

grated functional proteins and cell division.

C O L L A B O R AT I O N S International Igor KomarovShevchenko University, Kiev, UkraineMarina RautenbachStellenbosch University, Stellenbosch, South Africa

National Alfred BlumeMartin-Luther Universität Halle-Wittenberg, HalleSandro KellerTechnische Universität Kaiserslautern Michael KumkeUniversität PotsdamMartin SchulzeFNL, SchönowHeiko TraupeUniversitätsklinikum Münster 

Karl Sydow

Heike Nikolenko,

Kathi Scheinpflug

Margitta Dathe and

group members

110 111

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2R E S E A R C H G R O U P S / / / C H E M I C A L B I O L O G Y F O R S C H U N G S G R U P P E N / / / C H E M I S C H E B I O L O G I E

C H E M I C A L S Y S T E M S B I O L O G Y

C H E M I S C H E S Y S T E M B I O L O G I E

G R O U P L E A D E RD R . R O N A L D F R A N K

B I O G R A P H Y

1967 – 1973 Studied chemistry, biochemistry, and microbiology at the Universität Hamburg, Germany

1974 Diploma in chemistry at the Universität Hamburg, Germany

1975 – 1979 Ph.D. at the Universität Hamburg (Prof. Köster)

1979 – 1980 Post-Doc at the Institute of Organic Chemistry and Biochemistry with Prof. Köster at the Universität Hamburg

1980 – 1984 Post-Doc at Gesellschaft für Biotechnologische Forschung mbH (GBF) with Dr. Blöcker in Braunschweig, Germany

since 1985 Staff scientist at GBF (renamed 2005 to Helmholtz Centre for Infection Research, HZI)

1995 – 2003 Head of research group “Molecular Recognition” at GBF

2003 – 2010 Head of department of Chemical Biology at the HZI

since 2008 Coordinator of ChemBioNet national infrastructure initiative for academic research in chemical biology at the Leibniz-Institut für Molekulare Pharmakologie (FMP) in Berlin-Buch

since 2009 Coordinator of the European Research Infrastructure Initiative EU-OPENSCREEN at FMP

2010 – 2012 Acting coordinator of FMP´s Chemical Biology Section

since 2010 Head of Research Group on Chemical Systems Biology at the FMP

S U M M A R Y

The Chemical Systems Biology Group pursues the investigation of cellular targets with multiple/poly-regulatory functions. Our goal is to develop smart pharmacological tools and approaches towards systems interventions and treatments.The group is currently pursuing two research projects: investigating cellular protein net-works regulated by calmodulin, a central calcium sensor and regulator, and advancing chemical microarray technology. SC2 chemical microarray technology developed by the principal investigator is used to discover small molecule ligands of protein targets in group projects and is offered to internal and external cooperation partners. The group was home of the Chemical Biology Unit’s module “synthetic chemistry” which supports research projects through the chemical synthesis of custom-fit molecules.The coordinating office of EU-OPENSCREEN is also part of the group. The preparatory phase of this ESFRI infrastructure initiative, “European infrastructure of open screening platforms for Chemical Biology,” is funded by the EC and includes 21 partners in 14 Eu-ropean countries. This initiative was recently included by the German Ministry od Educa-tion and Research (BMBF) into the National Roadmap for large Research Infrastructures.

Z U S A M M E N FA S S U N G

Die Gruppe Chemische Systembiologie beschäftigt sich mit der Untersuchung zellulä-rer Zielmoleküle („Targets“) mit vielfachen regulatorischen Funktionen. Unser Ziel ist es, intelligente pharmakologische Werkzeuge und Lösungswege zu entwickeln, die in Krank-heitssysteme eingreifen und sie behandeln können.Die Gruppe verfolgt derzeit zwei Forschungsprojekte: die Untersuchung zellulärer Prote-innetzwerke, die durch Calmodulin, einen zentralen Calcium-Sensor und -Regulator, regu-liert werden, und das Voranbringen der chemischen Mikroarray-Technologie. Die vom Gruppenleiter entwickelte SC2-Methode zur Herstellung chemischer Mikroarrays wird eingesetzt, um in Projekten der Gruppe kleine molekulare Liganden von Protein-Targets zu entdecken. Die Mikroarrays werden auch Kooperationspartnern innerhalb wie außer-halb der Einrichtung zur Verfügung gestellt. Aus der Arbeitsgruppe ist das Modul „Syn-thetische Chemie“ des Bereichs Chemische Biologie hervorgegangen, das mittels che-mischer Synthese maßgeschneiderter Moleküle Forschungsprojekte unterstützt.Die Gruppe ist auch für die Koordination des europäischen Screeningprojekts EU-OPEN-SCREEN zuständig. Die Vorbereitungsphase dieser ESFRI-Infrastrukturinitiative “Euro-pean infrastructure of open screening platforms for Chemical Biology” wird von der Europäischen Kommission finanziert und hat 21 Partner in 14 europäischen Ländern. Das Vorhaben wurde kürzlich vom BMBF in die Deutsche Roadmap für große Forschungsin-frastrukturen aufgenommen.

D E S C R I P T I O N O F P R O J E C T S

Biological FocusCellular hub proteins with thousands of interaction partners or substrates such as the heat shock proteins, ribosomes, protea-somes, etc. are increasingly coming into focus as pharmacological targets for malignancies like cancer and rare diseases. Malignant cells are often dependent on such targets and are driven to apop-tosis when these are down regulated, while healthy cells tolerate significant deviations from normal activities.Stimulated by earlier work of the PI, we have chosen calmodulin (CaM) for our studies, which is an integral modulator of many calcium-dependent processes in virtually every eukaryotic cell type. CaM is a small acidic ubiquitous protein; it functions as a cytosolic calcium receptor and binds up to four calcium ions with very high affinity. CaM responds to a variety of extracellular signals which increase the cytosolic calcium level and it regulates many enzymes involved in signal transduction (Fig. 1, p. 112). There are also protein families including neuromodulin, myosin, and ion channels that bind CaM at remarkably low concentrations or even in the absence of calcium. CaM is also discussed as a translocation chaperone for small proteins. Our particular interest is to study the CaM-mediated regulation of ribosomal proteins (Fig. 2, p. 112). Ribosomal disruption is the cause of several hematopoietic disorders (Taylor AM et al.,Exp Hematol. 2012, 40: 228–237) and CaM has been implicated in downstream effects. By using a peptide array scan of all protein components of the human ribosome in the format of 21mer over-lapping peptide fragments (5000 peptides altogether, including reference peptides), we confirmed well reported CaM binding sites and also identified several new ones.

Chemistry FocusWe are exploring small molecular scaffolds for their utility as probes to modulate our target proteins. This work involves the chemical synthesis of such scaffolds, including small libraries of soluble compounds for complementation of the FMP chemical collection in regions of sparsely populated chemical space (e.g. tetra- and pentasubstituted dihydropyrroles, bi- and tricyclic lac-tames). Successful syntheses are then adapted to our special SC2 chemical microarray technology, followed by the preparation of combina-torial libraries, each about 2,500 analogues around such scaffolds.

These cellulose-conjugated libraries are printed in duplicate onto glass microscope slides and screened for binding to our target proteins. For CaM we have identified several calcium-dependent binders in our diketopiperazine (DKP) libraries. Selected binders were re-synthesized and modified for initial SAR studies. Their activity in the classical calcineurin assay revealed IC50 values down to the low picomolar range!

Synthetic Chemistry Module of the Chemical Biology Unit(Judith Holz and André Horatscheck)In many projects custom-fit molecules are essential and can only be provided by individual chemical synthesis. Lack of chemical expertise and capacity in this area can often delay a project or even terminate it. To provide a powerful support and a reliable partner, the chemistry module was installed in 2012. Its expertise comprises chemical synthesis as well as a background in medicinal chemistry. Therefore, it can work on chemical related questions like improvement of inhibitor activity, fluorescence labeling, op-timizing solubility, cell permeability, or simply in synthesizing a focused library of chemical compounds. Currently, the optimization of a Shp2 inhibitor in cooperation with Prof. Birchmeier from the MDC is a central project (PNAS 2008). Shp2 is a protein tyrosine phosphatase that acts mostly down-stream of receptor tyrosine kinases and upstream of the Ras/ERK pathway. Gain-of-function, as well as loss-of-function mutations have been identified in humans which lead to various develop-mental disturbances and cancer, making Shp2 a drug target of high importance. The other main project, in cooperation with AG Haucke, deals with the optimization of Pitstop 2, an inhibitor of clathrin-mediated en-docytosis. Pitstop 2 binds to the terminal domain of the clathrin heavy chain, thereby preventing coated pit assembly (Cell 2011). With the crystal structure of Pitstop 2 and the terminal domain of clathrin as a basis, a small focused library of compounds is being synthesized. In parallel a random library is synthesized and both will be tested to provide sufficient data for SAR studies.

Khalid Abu Ajaj,

Lioudmila Perepelittchenko

Edelgard Schmeisser,

Irina Nickeleit

112 113

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S G R U P P E N / / / C H E M I S C H E B I O L O G I E

S E L E C T E D P U B L I C AT I O N S Bieschke J, Herbst M, Wiglenda T, Friedrich RP, Boeddrich A, Schiele F, Kleckers D, Lopez del Amo JM, Grüning BA, Wang Q, Schmidt MR, Lurz R, Anwyl R, Schnoegl S, Fandrich M, Frank R, Reif B, Gunther S, Walsh DM, Wanker EE (2012) Small-molecule conversion of toxic oligomers to nontoxic beta-sheet-rich amyloid fibrils. Nature Chem Biol 8: 93-101.

Kopp K, Buntru A, Pils S, Zimmermann T, Frank R, Zumbusch A, Hauck CR (2012) Grb14 is a negative regulator of CEACAM3-mediated phagocytosis of pathogenic bacteria. J Biol Chem 287: 39158-39170.

Taft F, Harmrolfs K, Nickeleit I, Heutling A, Kiene M, Malek N, Sasse F, Kirschning A (2012) Combined muta- and semisynthesis: a powerful synthetic hybrid approach to access target specific antitumor agents based on ansamitocin P3. Chemistry 18: 880-886.

Grelle G, Otto A, Lorenz M, Frank R, Wanker EE, Bieschke J (2011) Black tea theaflavins inhibit formation of toxic amyloid-beta and alpha-synuclein fibrils. Biochemistry 50: 10624-10636.

Heinzelmann K, Scholz BA, Nowak A, Fossum E, Kremmer E, Haas J, Frank R, Kempkes B (2010) Kaposi’s sarcoma-associated her-pesvirus viral interferon regulatory factor 4 (vIRF4/K10) is a novel interaction partner of CSL/CBF1, the major downstream effector of Notch signaling. J Virol 84: 12255-12264.

FMP authorsGroup members

E X T E R N A L F U N D I N G European Union, European Infrastructure of Open Screening Platforms for Chemical Biology EU-OPENSCREEN, GA 261861, 11/2010 – 10/2013, 3.700.000 Euro

European Union, “Building data bridges between biological and medical infrastructures in Europe”, BioMedBridges, 01.2012 – 12.2015, 137.498 Euro

R E S E A R C H G R O U P S / / / C H E M I C A L B I O L O G Y

G R O U P M E M B E R S Dr. Irina Nickeleit *Dr. Khalid Abu Ajaj *Dr. Lioudmila Pereplittchenko (technical assistant)Edelgard Schmeisser (technical assistant)

Synthetic Chemistry Module of the Chemical Biology Platform Dr. Judith Holz *Dr. André Horatscheck *Sylvia Oestreich (technical assistant) *Sandra Miksche (technical assistant) *

EU-OPENSCREEN team Dr. Anne Höner (Project Manager)Dr. Bahne Stechmann (Scientific Manager) Martin McLean, MA (assistant project manager) Dr. Torsten Meiners (guest)

Group members as of 31.12.2012* Part of reporting period

Fig. 1: Our central target protein: Calcium-loaded calmodulin (CaM) in

the ligand free (top) state and in complex with a target peptide (bottom),

showing the large conformational changes of CaM during target binding

(taken from Hultschig et al., 2004, J. Mol. Biol. 343, 559-568).

The EU-OPENSCREEN coordination team:

Bahne Stechmann, Ronald Frank, Martin McLean, Anne Höner

Fig. 2: The images show the colocalization of

calmodulin and ribosomal proteins in Hela cells.

Top: merged images; middle: CaM localisation;

bottom: ribosomal protein localisation.

C O L L A B O R AT I O N S International EU-OPENSCREEN; 18 research institutes and 3 further organizations from 14 mem-ber states. The group actively supports the building of a European research infrastructure of screening platforms for Chemical Biology in the frame of the ESFRI roadmap.

Annie AndrieuxGrenoble-Institute des Neurosciences, Grenoble, FranceAndreas MeyerhansUniversity Pompeu Fabra, Barcelona, SpainLeonard ZonHoward Hughes Medical Institute, Boston, USA

National Hans-Joachim FritzVarignost, GöttingenGerhard HunsmannVarignost, GöttingenOle BrandtIntavis Bioanalytical Instruments, CologneChristian BehnIntavis Bioanalytical Instruments, CologneMartin SchwemmleInstitute of Virology, University of FreiburgErich WankerMax-Delbrück Centre for Molecular Medicine, BerlinBettina KempkesHelmholtz Centre Munich for Environmental Health, Munich Christoph HauckInstitute for Cell Biology, University of Konstanz

114 115

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2R E S E A R C H G R O U P S / / / C H E M I C A L B I O L O G Y F O R S C H U N G S G R U P P E N / / / C H E M I S C H E B I O L O G I E

M A S S S P E C T R O M E T R Y

M A S S E N S P E K T R O M E T R I E

G R O U P L E A D E RD R . E B E R H A R D K R A U S E

B I O G R A P H Y

1975 Diploma degree in physical chemistry at the Humboldt University, Berlin

1982 Dr. rer. nat., Humboldt University, Berlin

1984 – 1986 Research Group Leader ‘Drug Development’ in the Pharmaceutical Industry

1987 – 1991 Research Associate at the Institute of Drug Research

since 1992 Senior Scientist and Head of the Mass Spectrometry Group at the FMP

S U M M A R Y

Our group focuses on the development and application of mass spectrometry-based proteomic methods to investigate cellular signaling processes. Our main topics of re-search have been protein-protein interactions and post-translational modifications and their functional consequences. In this context we are interested in acetylation-dependent protein-protein interactions of histone H4 that might further modulate gene expression. Our results indicate that the H4 acetylation state establishes its regulatory effects in a cumulative manner rather than through a site-specific recruitment of regulatory proteins, which is not consistent with the “histone code” theory. A second project addresses T cell receptor-associated protein interactions which are mediated by reversible tyrosine phosphorylations and may function as modulators of cellular adhesion and migration processes.

Z U S A M M E N FA S S U N G

Wir beschäftigen uns vorwiegend mit der Entwicklung und Anwendung massenspekt-rometrischer Methoden zur Proteomanalyse, um zelluläre Signalwege zu untersuchen.Unsere zentralen Forschungsthemen sind Protein-Protein-Interaktionen und posttrans-lationale Modifikationen im Hinblick auf deren funktionale Auswirkungen. In diesem Zusammenhang interessieren wir uns für die acetylierungsabhängigen Protein-Protein-Interaktionen von Histon H4, die die Genexpression weiter modulieren könnten. Unsere Ergebnisse deuten darauf hin, dass der Acetylierungsstatus von H4 seine regulatorischen Wirkungen eher in additiver Weise ausübt als über positionsspezifische Interaktionen acetylierter Lysinsdeitenketten mit regulatorischen Proteine, was gegen die „Histon-Code”-Theorie spricht. In einem weiteren Projekt beschäftigen wir uns mit T-Zell-Rezep-tor-assoziierten Protein-Wechselwirkungen, die durch reversible Tyrosinphosphorylierun-gen vermittelt werden und als Modulatoren zellulärer Adhäsions- und Migrationsprozesse fungieren können.

D E S C R I P T I O N O F P R O J E C T S

Phosphorylation of T cell adapter proteins. T cell receptor stimulation is accompanied by multiple tyrosine phosphorylation of kinases and adapter proteins leading to acti-vation of integrins and to corresponding changes in the adhesive and migratory properties of the cell via nucleation of macromo-lecular signaling complexes. The adhesion and degranulation promoting adapter protein (ADAP) which is multiply phosphory-lated by T cell receptor activated kinases acts as an adapter for the formation of a receptor-proximal signaling complex. We use mass spectrometry to map tyrosine phosphorylation sites in ADAP comprehensively. The analysis reveals several sites of modification that comprise previously identified, as well as novel, sites. Two of these motifs are located in the folded hSH3 domains of ADAP at helix-sheet interfaces. The role of individual phosphotyrosines for protein complex formation and the regulation of cellular adhe-sion are still under debate. At first, we identified phosphorylation-dependent interaction partners binding to unstructured regions of ADAP using peptide pull-down assays. Phosphotyrosine pep-tide motifs covering Y571, Y595, Y625, and Y771 and the corre-sponding nonphosphorylated sequences were covalently coupled to agarose beads and incubated with Jurkat T cell lysates. For unambiguous differentiation between phosphorylation-specific and nonspecific protein interaction, we employed metabolic iso-tope labeling (SILAC) in combination with high-resolution mass spectrometry (Fig. 1, p. 116). In addition to previously known SH2 domain-based interactions of ADAP with SLP76, we identified novel ADAP interaction partners such as the Ras GTPase activat-ing protein and NCK which belong to the larger TCR proximal signaling complex. Subsequently, we studied the role of tyrosine-phosphorylations within the N-terminal hSH3 domain of ADAP to gain further insights into the role of structural domains in T cell signaling. Using site-specifically phosphorylated ADAP-hSH3N (at position Tyr 571) we performed pull-down experiments with Jurkat T cells and primary human T cells for identification of phospho-specific interaction partners. Quantitative mass spectrometry re-vealed very few phosphorylation-dependent interaction partners. All of these contain SH2 domains and are known to play a role in TCR signaling but are not known to interact directly with ADAP (e.g. ZAP70). NMR experiments and functional assays using YF mutants have been performed to confirm the direct interaction with site-specifically phosphorylated ADAP.

Novel proteomics approachTo achieve efficient and robust quantification of proteins in in-teractome analysis using primary cells ex vivo, we developed an alternative proteomic approach, as the widely-used metabolic la-beling method SILAC is impracticable (Fig. 2, p. 116). The shotgun approach employs two-dimensional RP-RP nanoLC for separation of tryptic peptides and 18O-labeling for relative quantification of proteins. Analyzing the phosphotyrosine interactome of ADAP, we demonstrated that the newly developed approach provides a high dynamic range as well as a limit-of-detection in the low fem-tomole sensitivity, allowing the identification of specific proteins in pull-down experiments.

Histone H4 acetylationHistone modifications play crucial roles in genome regulation with lysine acetylation being implicated in transcriptional control. We performed a proteome-wide investigation on the acetylation-dependent protein-protein interactions of the N-terminal tail of histone H4. Quantitative peptide-based affinity MS experiments based on the stable isotope labeling by amino acids in cell culture (SILAC) approach were used to determine the interactomes of H4 tails mono-acetylated at the four known acetylation sites K5, K8, K12 and K16 as well as a K5/K12 bis-acetylated and fully tetra-acetylated H4 tail. A set of 29 proteins was found to be enriched on the fully acetylated H4 tail, while no specific binders of the mono and bis-acetylated tails were detected. These observations agree with earlier reports indicating that the H4 acetylation state establishes its regulatory effects in a cumulative manner rather than via site-specific recruitment of regulatory proteins.

Michael Schümann,

Benno Kuropka

Heike Stephanowitz,

Sabine Anker

116 117

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S G R U P P E N / / / C H E M I S C H E B I O L O G I E

S E L E C T E D P U B L I C AT I O N S Broncel M, Krause E, Schwarzer D, Hackenberger CP (2012) The Alzheimer’s disease-related tau protein as a new target for chemi-cal protein engineering. Chemistry 18: 2488-2492.

Kashikar ND, Alvarez L, Seifert R, Gregor I, Jackle O, Beyermann M, Krause E, Kaupp UB (2012) Temporal sampling, resetting, and adaptation orchestrate gradient sensing in sperm. J Cell Biol 198: 1075-1091

Maritzen T, Zech T, Schmidt MR, Krause E, Machesky LM, Haucke V (2012) Gadkin negatively regulates cell spreading and motility via sequestration of the actin-nucleating ARP2/3 complex. Proc Natl Acad Sci USA 109: 10382-10387

Stephanowitz H, Lange S, Lang D, Freund C, Krause E (2012) Im-proved two-dimensional reversed phase-reversed phase LC-MS/MS approach for identification of peptide-protein interactions. J Proteome Res 11: 1175-1183

Büchse T, Horras N, Lenfert E, Krystal G, Korbel S, Schümann M, Krause E, Mikkat S, Tiedge M (2011) CIN85 interacting proteins in B cells-specific role for SHIP-1. Mol Cell Proteomics 10: M110 006239.

FMP authorsGroup members

R E S E A R C H G R O U P S / / / C H E M I C A L B I O L O G Y

G R O U P M E M B E R S Benno Kuropka (doctoral student) *Diana Lang (doctoral student) *Michael Schümann (technical assistant) Heike Stephanowitz (technical assistant)

Group members as of 31.12.2012* Part of reporting period

Fig. 1: Phosphotyrosine interactome of ADAP.

Results of SILAC-based pull-down experiments

with site-specifically phosphorylated ADAP se-

quences. Scatter plots of heavy/light and light/

heavy isotopic ratios of identified proteins

from pull-down experiments using Tyr 571 (A),

Tyr 595 (B), Tyr 625 (C), and Tyr 771 (D) ADAP

baits.

Fig. 2: Principle of 18O/2-D-LC/MS/MS ap-

proach for identification of specific proteins in

interactome analysis. Phosphorylated and the

corresponding non-phosphorylated peptides

or proteins were covalently coupled to agarose

beads and subsequently incubated with hu-

man T cell lysate. Tryptic protein digestion

and C-terminal 16O/18O-isotope labeling was

performed directly on-bead in the presence of

“light” and “heavy” labeled water. Peptides

were separated and identified by 2-D (RP-RP)-

nanoLC-MS/MS analysis.

C O L L A B O R AT I O N S International Miles HousleyUniversity of Glasgow, Scotland Remigiusz SerwaImperial College London, UK

National Bernd NürnbergEberhard Karls Universität TübingenU. Benjamin KauppCenter of Advanced European Studies and Research, BonnKurt EngelandUniversität Leipzig

Hans G. BörnerHumboldt-Universität zu BerlinVolker HauckeFreie Universität Berlin and FMP Petra KnausFreie Universität Berlin Christian HackenbergerFreie Universität Berlin and FMPChristian FreundFMP and Freie Universität Berlin Dirk SchwarzerFMP and Eberhard Karls Universität Tübingen

Michael Schümann Heike Stephanowitz

118 119

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2R E S E A R C H G R O U P S / / / C H E M I C A L B I O L O G Y F O R S C H U N G S G R U P P E N / / / C H E M I S C H E B I O L O G I E

S C R E E N I N G U N I T

G R O U P L E A D E RD R . J E N S P E T E R V O N K R I E S

B I O G R A P H Y

1987 Diploma in biology at the University Hospital Hamburg-Eppendorf (Prof. Strätling)

1991 Ph.D. at the University Hospital Hamburg-Eppendorf (Prof. Strätling)

1995 – 2000 Research on proteins involved in origin of cancer in the lab of W. Birchmeier

2000 – 2003 Head of Screening Unit of Semaia Pharmaceuticals

since 2003 Head of Screening Unit at FMP

2011 Chairman Gemeinsame Fachgruppe Chemische Biologie DECHEMA

2012 Advisory Board SFICAST (University Oslo)

since 2012 Head of the joint study section for chemical biology of DPhG, GBM, GDCh, and DECHEMA

S U M M A R Y

The Unit serves as an open access technology platform for automated screening, using either compound libraries such as the ChemBioNet collection (35,000 cpds) or genome-wide RNAi libraries (human, mouse, nematodes). Beside supporting assay development, process automation, screening and automated data analysis, the Unit identifies novel screening technologies (such as impedance measurements, High-Content-Screening, AlphaScreen, capillary electrophoresis, real-time kinetic cell-based assays) and imple-ments these for service. Three modules of the Unit support compound screening proj-ects: Screening module (Silke Radetzki), Compound management (Edgar Specker), and Process Automation. (Martin Neuenschwander). Compound management with auto-mated freezers currently provides a storage capacity for more than 200,000 probes. Furthermore, this module collects unique compounds donated from academic chemists and monitors quality through mass spectroscopy analysis. Genome-wide RNAi screening (Katina Lazarow) has been established as an additional service module complementing the identification of cellular targets by demonstrating the generation of similar cellular phenotypes by either compounds or through RNA-interference.

Z U S A M M E N FA S S U N G

Die Screening-Unit dient als frei zugängliche Technologieplattform für automatisierte Wirkstoffsuchen im Hochdurchsatz („Screenings“); verwendet werden entweder Subs-tanzbibliotheken wie die ChemBioNet-Sammlung (35.000 chemische Substanzen) oder genomweite RNAi-Bibliotheken (Mensch, Maus, Nematoden). Neben der Unterstützung in der Testentwicklung, Prozessautomatisierung, dem Screening und der automatischen Datenanalyse identifiziert die Unit neue Screening-Techniken (wie Impedanzmessungen, High-Content-Screening, AlphaScreen, Kapillarelektrophorese, zellbasierte Tests mit Echtzeit-Kinetik) und implementiert diese für den Routineeinsatz. Drei Module der Einheit unterstützen derzeit Substanz-Screeningprojekte: Screening-Modul (Silke Radetzki), Sub-stanzverwaltung (Edgar Specker) und Prozessautomatisierung (Martin Neuenschwander). Die Substanzverwaltung in automatisierten Gefrierkammern bietet derzeit eine Lagerka-pazität für mehr als 200.000 Proben. Außerdem integriert dieses Modul einzigartige Sub-stanzen in seine Sammlung, die von Wissenschaftlern aus nichtkommerziellen Institutio-nen beigesteuert wurden, und überwacht deren Qualität mittels massenspektroskopischer Analyse. Das genomweite RNAi-Screening (Katina Lazarow) wurde als Service-Einheit eta-bliert, um zelluläre Zielstrukturen („Targets“) durch die Erzeugung ähnlicher zellulärer Phä-notypen entweder durch chemische Substanzen oder RNA-Interferenz zu identifizieren.

D E S C R I P T I O N O F P R O J E C T S

Interfering with Influenza infection by inhibition of host cell functionsTargeting of host cell functions like enzymatic activities repre-sents a novel therapy concept against viral infection developed by Thomas Meyer (Max-Planck for Infection Biology, Berlin). In col-laboration with the EU-AntiFlu consortium (coordinator: Thomas F. Meyer), we have started to validate inhibitors of kinases and phosphatases, which had been previously identified by genome-wide RNA-interference to play key roles in viral infection. Primary hits identified by employing a capillary electrophoresis screen with the kinase CLK1 have already been confirmed to interfere with viral growth in host cells.

First inhibitors for MALT-1 caspase interfering with NFKB signaling in B cell lymphoma (ABC-DLBCL)The Screening Unit served for HTS with the ChemBioNet collec-tion using MALT-1 caspase (Nagel et al. 2012, Cancer Cell) and a FRET assay using a synthetic peptide as the substrate for the pro-tease. The collaboration with the laboratory of Daniel Krappmann (Helmholtz Zentrum München) resulted in identification of inhibi-tors of MALT-1, which selectively kill diffuse-large B cell lymphoma (ABC-DLBCL) in vitro and in vivo. As these compounds are already widely used as antipsychotic drugs, they will facilitate clinical trials for an off-label use in ABC-DLBCL therapy.

Specific inhibition of Clathrin mediated endocytosisCellular transport from the cell’s outside border to intracellular compartments, mediated by membrane vesicles and the protein clathrin, plays a key role in the entry of pathogens (HIV, bacteria), but also in the nervous system for synaptic signaling, and for re-cycling of trans-membrane receptors. In collaboration with the research group of Volker Haucke (FU & FMP, Berlin) we identified compounds named ‘pitstops’, which specifically interfere with clathrin protein interactions and thereby also interfere with HIV infection (von Kleist et al. 2011, Cell). For identification of inhibi-tors we used an ELISA for quantification of clathrin to amphiphy-sin binding.

Wnt-signaling inhibitors interfering in vitro and in vivo with growth of tumor cells Canonical Wnt signaling is deregulated in several types of human cancer, where it plays a central role in tumor cell growth and progression. The last step of the signal cascade consists of gene activation by b-Catenin in complex with LEF/TCF transcription factors. In collaboration with Stefan Krauss (University of Oslo) we set up a High-Content Screen with automated microscopes using a GFP reporter cell line, which produces Green Fluorescent Protein under the control of endogenous b-Catenin and TCF com-plexes. The Screening Unit identified two novel small molecules that specifically inhibit the canonical Wnt pathway at the level of the destruction complex for b-Catenin, which becomes deactivat-ed by mutations in many human tumors (Cancer Research, 2011). Specificity of compounds was verified in vitro using various cellular reporter systems and gene expression profile analysis. Tankyrase was identified as the target of the inhibitor, which acts through stabilization of Axin2 a negative regulator of Wnt signaling (Can-cer Research, 2012). The compounds were also validated in vivo using the Xenopus double-axis formation assay, by inhibiting the growth of human tumor cells in mouse xenografts, and in ApcMin mice (multiple intestinal neoplasia, Min), which represent an ani-mal model for the development of human colon cancer.

Inhibitors for Met-receptor mediated metastasis of tumor cellsGrowth of tumors at distant sites (metastasis) is the process respon-sible for over 90% of cancer deaths. The Screening Unit focused its research on the inhibition of Met-receptors-induced scattering of tumor cells in vitro as a cellular model for metastasis. A cellular assay with a human pancreatic tumor cell line was optimized for high content screening with automated microscopes in 384 well format using fluorescent staining of DNA, of actin filaments and cytoplasm. Automated object identification of colonies (unscat-tered, –HGF) and scattered cells (+HGF) was set up on the basis of average distance calculations for neighboring cells (MolDiaP-acra, EU funded, SFMET, EU funded). Moreover, we established a label-free impedance measurement procedure for scattering. While imaging allowed us to identify Met-induced scattering in the hours range, the impedance measurements identified signifi-cant alterations on a scale of minutes.

Martin NeuenschwanderKaty Franke,

Kevin Mallow,

Edgar Specker

120 121

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S G R U P P E N / / / C H E M I S C H E B I O L O G I E

S E L E C T E D P U B L I C AT I O N S Habermann K, Mirgorodskaya E, Gobom J, Lehmann V, Müller H, Blümlein K, Deery MJ, Czogiel I, Erdmann C, Ralser M, von Kries JP, Lange BM (2012) Functional analysis of centrosomal kinase substrates in Drosophila melanogaster reveals a new function of the nuclear envelope component otefin in cell cycle progression. Molecular and cellular biology 32: 3554-3569.

Nagel D, Spranger S, Vincendeau M, Grau M, Raffegerst S, Kloo B, Hlahla D, Neuenschwander M, Peter von Kries J, Hadian K, Dorken B, Lenz P, Lenz G, Schendel DJ, Krappmann D (2012) Pharmaco-logic Inhibition of MALT1 Protease by Phenothiazines as a Thera-peutic Approach for the Treatment of Aggressive ABC-DLBCL. Cancer Cell 22: 825-837.

Waaler J, Machon O, Tumova L, Dinh H, Korinek V, Wilson SR, Paulsen JE, Pedersen NM, Eide TJ, Machonova O, Gradl D, Vo-ronkov A, von Kries JP, Krauss S (2012) A novel tankyrase inhibitor decreases canonical Wnt signaling in colon carcinoma cells and reduces tumor growth in conditional APC mutant mice. Cancer Res 72: 2822-2832.

Due AV, Kuper J, Geerlof A, von Kries JP, Wilmanns M (2011) Bisubstrate specificity in histidine/tryptophan biosynthesis isom-erase from Mycobacterium tuberculosis by active site metamor-phosis. Proc Natl Acad Sci USA 108: 3554-3559.

Timm T, von Kries JP, Li X, Zempel H, Mandelkow E, Mandelkow EM (2011) Microtubule affinity regulating kinase activity in living neurons was examined by a genetically encoded fluorescence resonance energy transfer/fluorescence lifetime imaging-based biosensor: inhibitors with therapeutic potential. J Biol Chem 286: 41711-41722.

von Kleist L, Stahlschmidt W, Bulut H, Gromova K, Puchkov D, Robertson MJ, MacGregor KA, Tomilin N, Pechstein A, Chau N, Chircop M, Sakoff J, von Kries JP, Saenger W, Krausslich HG, Shupliakov O, Robinson PJ, McCluskey A, Haucke V (2011) Role of the clathrin terminal domain in regulating coated pit dynamics revealed by small molecule inhibition. Cell 146: 471-484.

Waaler J, Machon O, von Kries JP, Wilson SR, Lundenes E, Wedlich D, Gradl D, Paulsen JE, Machonova O, Dembinski JL, Dinh H, Krauss S (2011) Novel synthetic antagonists of canoni-cal Wnt signaling inhibit colorectal cancer cell growth. Cancer research 71: 197-205.

FMP authorsGroup members

E X T E R N A L F U N D I N G Bundesministerium für Bildung und Forschung (BMBF), Helmholtz Initiative für Wirkstoffforschung, Chemical Biology Platform, coop-eration of MDC and FMP, funding of infrastructure and personnel, 2011 – 2015, 2.200.000 Euro (The institutional continuation as long-term cooperation is in-tended)

Deutsche Forschungsgemeinschaft, FOR 806, Z1, “Synthesis, op-timisation, and screening of small molecule libraries targeting protein-protein interactions”, RA 895/5-1, with J. Rademann, M. Beyermann, 05.2007 – 05.2010, 130.814 Euro

Bundesministerium für Bildung und Forschung (BMBF), “Screen-ing Unit: Assay development screening for lead identification and optimization”, 01GU0514 – KR, with C. Freund, R. Kühne, 01.2006 – 05.2009, 543.976 Euro

Europäische Kommission, 6. Framework Programme, „New screening techniques and strategies in the early diagnosis of solid tumours“, PL018771, with J. Rademann, 08.2006 – 01.2010, 400.006 Euro

European Union, 7. Framework Programme, EU-ANTIFLU FP7, “Innovative anti-influenza drugs excluding viral escape”, Co-ordinator: Thomas F. Meyer (MPI-IB, Berlin), 06.2011 – 05.2015, 400.000 Euro

European Union, 7. Framework Programme, EU-SFMET FP7-HEALTH-2007-A, “HGF/SF and MET in metastasis”, Coordina-tor: Ermanno Gherardi, (MRC-Cambridge, UK), 04.2008 – 03.2011, 223.750 Euro

Leibniz-Gemeinschaft, SAW, PAKT (FLI-Jena), with W. Rosenthal, 01.2011 – 12.2012, 180.000 Euro

Charité-ECRC Verbundprojekt, with MDC, 01.2009 – 06.2012, 414.000 Euro

Bundesministerium für Bildung und Forschung (BMBF), BMBF/MDC, RNAi & REMP, with MDC, with J. Rademann, 11.2008 – 2009, 800.000 Euro

Bundesministerium für Bildung und Forschung (BMBF), NGFN-plus, 5 Projects funded, 06.2008 – 05.2013, 150.000 Euro

R E S E A R C H G R O U P S / / / C H E M I C A L B I O L O G Y

G R O U P M E M B E R S Dr. Simone Gräber (High Content Screening) *Dr. Katina Lazarow (RNAi) *Dr. Martin Neuenschwander (Process Automation, HTS-Analysis)Dr. Silke Radetzki (High Content Screening)Dr. Edgar Specker (Compound Management, Chemistry)Dipl. Biol. Jamina Eckhard (RNAi) *M.Sc. Marc Wippich (HTS) *Chris Eckert (technical assistant) *Katy Franke (technical assistant)Sabrina Kleißle (technical assistant)Kevin Mallow (technical assistant)Andreas Oder (technical assistant) *Carola Seyffarth (technical assistant)

(HTS: high throughput screening)Group members as of 31.12.2012* Part of reporting period

Fig. 1: We set up a 2D cell culture assay for high content screening with

automated microscopes, which represents a model for metastasis of tumor

cells initiated by addition of Hepatocyte Growth Factor (A, C: no initiation

of metastasis, B, D: initiation) and enables one to track the migration

of about 2,000 cells by colored lines for 24 hours (C,D) using in-house

developed software. Without initiation, cells build colonies contacting each

other and migrating within the colony area (A, C). After initiation, cells lose

contact and migrate out of the colony area (B, D). Tumor cells are fluores-

cently stained red for actin (phalloidin) and green-blue for nuclei (Hoechst

3342, CMFDA).

Fig. 2: In the absence of an inhibitor, b-Catenin (fluorescence) mainly local-

izes in the nuclei of colon cancer cells (left image), while application of the

compound blocks nuclear localization and b-Catenin localizes at cellular

borderlines in adherens junctions (right image).

The compound has been shown to inhibit tankyrase enzyme activity and

thereby increases axin protein level in cells. Axin stimulates degradation of

b-Catenin protein, which is not protected by cadherin protein in the adher-

ens junctions, thus preventing Catenin signaling in the nucleus.

C O L L A B O R AT I O N S International Stefan KnappOxford University, UK Ermanno GherardiUniversity Pavia , ItalyTom BlundellMRC, Cambridge, UK Stefan KraussUniversity Oslo, Norway

National Thomas F. MeyerMax-Planck-Institute for Infection Biology, BerlinWalter BirchmeierMax-Delbrück Center for Molecular Medicine, BerlinThomas GressUniversitätsklinikum, MarburgClemens SchmittCharité – Universitätsmedizin Berlin

Silke Radetzki,

Jens Peter von Kries,

Carola Seyffarth

Katy Franke

122 123

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2R E S E A R C H G R O U P S / / / C H E M I C A L B I O L O G Y F O R S C H U N G S G R U P P E N / / / C H E M I S C H E B I O L O G I E

P R O T E I N C H E M I S T R Y

P R O T E I N C H E M I E

G R O U P L E A D E RP R O F. D R . D I R K S C H W A R Z E R

B I O G R A P H Y

1999 – 2002 PhD in Chemistry at the Philipps-Universität in Marburg with Mohamed A. Marahiel

2003 – 2006 Postdoctoral research with Philip A. Cole at the Johns-Hopkins University, Baltimore, USA

2006 – 2007 Research stay with Henning D. Mootz at the Universität Dortmund

2007 – 2011 Emmy-Noether Group leader at the Leibniz-Insititut für Molekulare Pharmakologie (FMP) in Berlin

since 2011 Professor of Biochemistry at the Interfaculty Institute of Biochemistry (IFIB), University of Tübingen

S U M M A R Y

All eukaryotes organize their genomes in the form of chromatin, a complex of DNA and dedicated packing proteins, the histones. In addition to compacting DNA, chromatin regulates the activity of encoded genes through posttranslational modifications of his-tone proteins. Deciphering the complex crosstalk between histone modifications and gene activity represents a central challenge for biomedical science. Our group is devel-oping and using chemical tools to study the physiological functions of posttranslational histone modifications. We combine methods from chemistry, biochemistry and molecular biology to probe a wide range of chromatin factors, including chromatin binding proteins and histone-modifying enzymes (Fig. 1, p. 124).

Z U S A M M E N FA S S U N G

Bei allen Eukaryoten ist das Genom in Form von Chromatin, einem Komplex aus DNA und zugehörigen Verpackungsproteinen, den Histonen, organisiert. Neben der Verpackung der DNA reguliert Chromatin die Aktivität der kodierten Gene durch posttranslationale Modifikationen der Histon-Proteine. Die Untersuchung der komplexen Wechselwirkungen zwischen Histon-Modifikationen und Genaktivität stellt eine zentrale Herausforderung für die biomedizinische Wissenschaft dar. Unsere Gruppe entwickelt und nutzt chemische Werkzeuge, um die physiologischen Funktionen posttranslationaler Histon-Modifikatio-nen zu erforschen. Wir kombinieren Methoden der Chemie, Biochemie und Molekular-biologie, um ein breites Spektrum an Chromatin-Faktoren, u.a. Chromatin-Bindungspro-teine und Histon-modifizierende Enzyme, zu untersuchen (Abb. 1, S. 124).

D E S C R I P T I O N O F P R O J E C T S

Histone Acetylation Reversible acetylation of lysine residues constitutes an important histone modification and is generally associated with active gene transcription (Fig. 2, p. 124). Over 20 acetylation sites have been identified on histone proteins to date. The acetylation level at indi-vidual lysine residues is determined by the balanced action of his-tone acetyl-transferases (HATs) and histone deacetylases (HDACs). HDACs have gained considerable attention recently because they have been identified as promising drug targets. Therapeutic inter-ventions of aberrant HDAC activities, which are believed to result in the down-regulation of pro-apoptotic genes in cancer, depend on the discovery of new drugs targeting HDACs. HDAC-related drug discovery relies on sophisticated assays that report HDAC activity by simple colorimetric or fluorogenic read-outs which can be adapted to high-throughput formats. We have established a simple and general synthesis procedure for colori-metric HDAC substrates by solid-phase peptide synthesis (SPPS).To this end, and in collaboration with Michael Beyermann (Peptide Synthesis, FMP), we devleloped a protocol for racemization-free couplings of amino acid chlorides that are able to form amide bonds with un-reactive amines of chromophores, which are used to report the HDAC activity. We could demonstrate the appli-cability of these substrates in the most commonly used type of HDAC assays. In contrast to conventional synthesis strategies, the approach established here allows for great flexibility in designing HDAC substrates with respect to the amino acids flanking the acetylation sites. Furthermore, our new substrates are not prone to a very prominent assay artifact of conventional HDAC assays (Dose et al. Chem Comm 2012). In collaboration with Philipp Selenko (In-cell NMR, FMP) we have further developed a method for monitoring histone deacetylation and acetylation reactions at multiple sites in real time. This ap-proach is based on specific NMR characteristics that lysine res-idues display upon acetylation: in 15N-HSQC experiments the backbone amide resonance of Lys residues experiences a chemi-cal shift upon acetylation and the unique signal of the Ne amide affords a new signal. Both signals can be used for monitoring acetylation and deacetylation reactions. Our approach includes a chemical synthesis scheme for the incorporation of stable, NMR-active isotopes at selected lysine positions within synthetic his-tone tails (Dose et al. ACS Chem Biol 2011).

Histone Phosphorylation Phosphorylation of serine and threonine residues is another long-known histone modification (Fig. 2, p. 124). In contrast to acetyla-tion and methylation of Lys residues, the physiological function of histone phosphorylation is only poorly understood and is puz-zling because these modifications are associated with condensed inactive chromatin and with relaxed and transcriptionally active chromatin region. We address this ambiguity in collaboration with Eberhard Krause (Mass Spectrometry, FMP) using mass spectrom-etry. According to the “histone code theory” histone modifica-tions constitute recognition sites for dedicated binding proteins which are recruited to chromatin. In order to identify these binding proteins we synthesize suitable proteomic baits that allowed the isolation of such binding proteins from cellular extracts (Klingberg et al., manuscript in preparation).

Evolved Sortase A Mutants for Histone Semisynthesis We have further developed and assessed an engineered sor-tase A for histone semisynthesis in collaboration with Christian Freund (Protein Engineering, FMP) and Michael Beyermann (Pep-tide Synthesis, FMP). Sortases have recently entered center-stage as protein engineering tools since they allow the chemoselective ligation of peptides and proteins. This process requires an LPxTG sorting motive in one of the peptides, which serves as a recogni-tion and cleavage site for the enzyme. Instead of being hydro-lyzed the enzyme bound intermediate is ligated to an N-terminal Gly residue of the second peptide resulting in a stabile peptide bond linking both ligation partners. The strict requirement for the LPxTG sorting-motive is one of the few downsides of sortase-me-diated ligations, which we have addressed in this project. Phage-display screening of a self-ligating sortase mutant library that was randomized in the predicted substrate recognition loop allowed us to isolate sortase mutants that ligate motives other than the canonical LPxTG sequence. One of these sortase mutants (F40-sortase) possessed a modified substrate selectivity suitable for traceless semisynthesis of histone H3, rendering the F40-sortase a powerful tool for chemical biology and chromatin biochemistry (Piotukh et al., JACS 2011).

Falko Schirmeister

(intern),

Rebecca Klingberg,

Jan Oliver Jost

124 125

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S G R U P P E N / / / C H E M I S C H E B I O L O G I E

S E L E C T E D P U B L I C AT I O N S Broncel M, Krause E, Schwarzer D, Hackenberger CPR (2012) The Alzheimer’s disease related tau protein as a new target for chemi-cal protein engineering. Chemistry 18: 2488-2492. Dose A, Jost JO, Spiess AC, Henklein P, Beyermann M, Schwarzer D (2012) Facile synthesis of colorimetric histone deacetylase sub-strates. Chem Comm (Camb) 48: 9525-9527. Liokatis S, Stützer A, Elsässer SJ, Theillet FX, Klingberg R, van Rossum B, Schwarzer D, Allis CD, Fischle W, Selenko P (2012) Phosphorylation of histone H3 Ser10 establishes a hierarchy for subsequent intramolecular modification events. Nat Struct Mol Biol 19: 819-823. Dose A, Liokatis S, Theillet FX, Selenko P, Schwarzer D (2011) NMR profiling of histone deacetylase and acetyl-transferase activi-ties in real time. ACS chemical biology 6: 419-424. Piotukh K, Geltinger B, Heinrich N, Gerth F, Beyermann M, Freund C, Schwarzer D (2011) Directed evolution of sortase A mutants with altered substrate selectivity profiles. Journal of the American Chemical Society 133: 17536-17539.

FMP authorsGroup members

E X T E R N A L F U N D I N G Deutsche Forschungsgemeinschaft, „Entwicklung und Nutzung chemischer Werkzeuge zur Untersuchung von Histon-Modifi-kationen und den zugehörigen Enzymen“, SCH 1163 /3-1, since 08.07, 930.000 Euro Deutsche Forschungsgemeinschaft, SPP 1623: Chemoselektive Reaktionen für die Synthese und Anwendung funktionaler Proteine, „Establishing a Chemical Tool Box for Modified Nucleosomes and its Application for Analysis of the Histone Code“, SCH 1163/4-1, since 08.12, 190.000 Euro

R E S E A R C H G R O U P S / / / C H E M I C A L B I O L O G Y

G R O U P M E M B E R S Alexander Dose (doctoral student) *Jan Oliver Jost (doctoral student) *Dr. Rebecca Klingberg (doctoral student, PhD in 2012) *Till Teschke (doctoral student) *Bernhard Geltinger (technical assistant) *

* Part of reporting period

Fig. 1: Overview of the group activities: The

chemical synthesis of modified histone tails is an

enabling tool used for a wide range of biochemi-

cal applications including proteomics, assay de-

velopment, probing protein-protein interactions

and protein semisynthesis.

Fig. 2: The structure of the nuclosome: H2A, red;

H2B, yellow; H3, green; H4, blue; DNA, gray

(top), and common histone modifications on the

N-terminal tails: phosphorylation, acetylation and

methylation (bottom)

C O L L A B O R AT I O N S International Michael S. CosgroveSyracuse University, New York, USA Guy LippensInstitut Pasteur de Lille, Lille, FranceValerio OrlandoDulbecco-Telethon-Institut, Rome, Italy

National Wolfgang FischleMPI Göttingen Christian HackenbergerFreie Universität Berlin *Philipp SelenkoLeibniz-Institut für Molekulare Pharmakologie (FMP)Eberhard KrauseLeibniz-Institut für Molekulare Pharmakologie (FMP)Christian FreundLeibniz-Institut für Molekulare Pharmakologie (FMP)Michael BeyermannLeibniz-Institut für Molekulare Pharmakologie (FMP)

* now at the FMP

Alexander Dose

Diana Lang

126 127

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2 I N D E XA D M I N I S T R AT I V E A N D T E C H N I C A L S E R V I C E S

I N D E X

D I R E C T O R AT E Prof. Volker Haucke Director since 01.01.2012 Prof. Hartmut Oschkinat Acting Director until 31.12.2011 Dr. Henning Otto Scientific Coordinator Katharina Schulz Scientific Coordinator Dr. Janet Zapke Scientific Coordinator Silke Oßwald Public Relations Manager Dr. Anne Höner EU-Liaison Officer Dr. Maxine Saborowski PhD-Programme Coordinator Dr. Birgit Oppmann Technology Transfer Dr. Adriane MenßenProject Manager “Wirtschafft trifft Wissenschaft” Alexandra Chylla Secretary Heidemarie Petschick Secretary A D M I N I S T R AT I O N Frank SchillingHead Administration Thomas EllermannGeneral Administration Silvia MauksPersonnel Management Marina SporsPersonnel Management Christel OttoGeneral Administration Claudia MessingGeneral Administration Mathias SchmidtGeneral Administration Renate WeimarGeneral Administration Gabriele SchumacherSecretary

AAbel, Sabine ............................................104Abu Ajaj, Khalid ......................................112Agrawei, Divya ........................................100Akbey, Ümit ...............................................36Al-Yamori, Raed ........................................48Albert, Gesa ..............................................40Albrecht, Sebastian ..................................68Artner, Lukas ............................................100

BBacetic, Jelena ..........................................76Backhaus, Carolin .....................................68Ball, Linda ..................................................36Ballaschk, Martin .......................................52Baranovic, Jelena ......................................84Bardiaux, Benjamin ...................................36Barone, Matthias .......................................48Beerbaum, Monika ...................................52Bekei, Beata ..............................................60Bellmann, Christian ...................................72Bender, Franziska ......................................80Bengtsson, Luiza .......................................68Bergsdorf, Eun-Yeong ..............................68Bertran, Jordi ..........................................100Beyermann, Michael ...............................102Billig, Gwendolyn ......................................68Binolfi, Andres ...........................................60Blasig, Ingolf E. .........................................70Blasig, Rosel ..............................................72Branz, Katharina ........................................76Breitkreutz-Korff, Olga .............................72Breng, Ingo..............................................126

CCarbone, Anna ..........................................84Castro Villela, Victor Manuel ....................72Chebli, Miriam...........................................84Chowdhury, Anup .....................................36Chylla, Alexandra ....................................126Claßen, Gala .............................................76Cording, Jimmi .........................................72Cremer, Nils ...............................................36Cruz e Silva, Andreia ................................68

DDabrowski, Sebastian ...............................72Dathe, Margitta .......................................106de Palma, Gregorio Guiseppe.................36Del Vecchio, Giovanna .............................72Diehl, Anne................................................36Diesenberg, Katrin ....................................76Döpfert, Jörg.............................................56Dorn, Matthias ..........................................52Dose, Alexander .....................................124Dreißigacker, Marianne ..........................126

EEckert, Chris ............................................120Eckhard, Jamina ......................................120Ehrlich, Angelika .....................................104Eichhorn-Grünig, Marielle ........................76Eichhorst, Jenny ........................................92Eichner, Miriam ...................................44, 72Eilemann, Barbara ....................................72Eisenmenger, Frank ..................................36Ellermann, Thomas .................................126Erdmann, Natalja ......................................36

FFeutlinske, Fabian .....................................76Fidzinski, Pawel .........................................68Fink, Uwe ...................................................76Frank, Ronald ..........................................110Franke, Katy .............................................120Franks, Trent ..............................................36Freund, Christian ......................................38Furkert, Jens ......................................88, 126

GGajera, Chandresh ....................................68Gehne, Nora .............................................72Geltinger, Bernhard ................................124Ghisi, Valentina .........................................84Gimber, Niclas ...........................................76Gödde, Kathrin .........................................68Göritz, Petra ..............................................68Goyette, Sandy .........................................60Gräber, Simone .......................................120

Gras, Claudia .............................................76Grimes, Andrew ......................................100Grzesik, Paul ..............................................44Günther, Ramona ......................................72Günther, Sebastian ...................................40

HHaas, Ann-Karin ........................................44Hackenberger, Christian P. R. ...................98Hahn, Sabine .............................................76Handel, Lilo ...............................................36Haseloff, Reiner .........................................72Haucke, Volker ................................4, 20, 74Heidenreich, Matthias ..............................68Heinrich, Nadja .......................................104Helmbrecht, Tolga ....................................52Helms, Hans-Christian ..............................72Hermann, Ingrid ......................................126Heyne, Alexander ...................................126Hiller, Matthias ..........................................36Hinz, Katrin M. ...........................................44Hoegg-Beiler, Maja ...................................68Hohensee, Svea ........................................92Holz, Judith .............................................112Höner, Anne ....................................112, 126Hoppmann, Christian .............................104Horatscheck, André ................................112Hoyer, Inna ................................................44Hube, Bianca .............................................72

JJabs, Sabrina .............................................68Jakob, Burkhard ........................................76Jäckel, Ronald .........................................126Jahn, Thomas ..........................................126Jayapaul, Jabadurai ..................................56Jentsch, Thomas J. ...............................8, 66Jost, Jan Oliver .......................................124Jurk, Marcel ...............................................52

KKaempf, Natalie ........................................76Kahlich, Bettina .........................................88

C O M P U T E R S E R V I C E S Thomas Jahn Head Network Administration until 30.09.2012 Ronald Jäckel Head Network Administration since 01.10.2012 Ingrid HermannSystem Administration Ingo Breng Service Engineer Alexander Heyne O F F I C E S Andrea Steuer Department of NMR-supported Structural Biology Marianne Dreißigacker Department of Chemical Biology Dr. Dietmar Zimmer Scientific Coordinator Department of Physiology and Pathology of Ion Transport Dr. Norma Nitschke Scientific Coordinator Department of Physiology and Pathology of Ion Transport A N I M A L FA C I L I T Y Dr. Elvira Rohde Head Animal Facility Eva Lojek Animal Care Keeper Lisa Prütz Animal Care Keeper W O R K P L A C E S A F E T Y Dr. Jens Furkert G E N E T I C E N G I N E E R I N G / B I O L O G I C A L S A F E T Y Dr. Ralf Schülein T E C H N I C A L S E R V I C E S Hans-Jürgen Mevert Marco Mussehl Holger Panzer Michael Uschner Stephanie Wendt Roy Wolschke Pascal Schulz

A D M I N I S T R A T I V E A N D T E C H N I C A L S E R V I C E S

128 129

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2I N D E X I N D E X

Kaiser, Hermann-Josef .............................68Kamdem, Nestor.......................................36Katchan, Ljudmila .....................................84Kath, Christina ...........................................76Kinne, Anita ...............................................44Klein, Wolfgang ........................................88Kleinau, Gunnar ........................................44Kleißle, Sabrina .......................................120Klingberg, Rebecca ................................124Klippel, Stefan .....................................40, 56Klippenstein, Viktoria ...............................84Klose, Annerose ......................................104Koch, Peter ................................................76Kochlamazashvili, Gaga ...........................76Kononenko, Natalia ..................................76Koo, Seong Joo ........................................76Korotkova, Tatiana ....................................78Kosslick, Daniela .......................................40Kosten, Jonas ............................................60Krause, Dagmar ..............................100, 104Krause, Eberhard ....................................114Krause, Gerd .............................................42Krauß, Michael ..........................................76Kreuchwig, Annika ....................................44Kries, Jens Peter von ..............................118Krönke, Nicole ..........................................68Krylova, Oxana ........................................108Kublik, Anja ...............................................72Kühne, Ronald ...........................................46Kunert, Britta .............................................36Kunth, Martin ............................................56Kuropka, Benno ......................................117

LLampe, André ...........................................76Lang, Diana .............................................117Lange, Sascha ...........................................36Lazarow, Katina .......................................120Leben, Rainer ............................................68Lehmann, Martin .......................................76Lehmann, Roland ......................................40Leidert, Martina ........................................36Leisle, Lilia .................................................68Lichtner, Gregor ........................................76Liebold, Janet ...........................................68

Rohde, Elvira ...........................................126Rose, Honor May ......................................60Rossa, Jan ..................................................72Rossella, Frederica ....................................56Rupp, Bernd ..............................................48Rutz, Claudia .............................................88

SSaborowski, Maxine ................................126Salazar, Hector ..........................................84Santos de Freitas, Monica ........................36Sargent, Catherine....................................44Scheinpflug, Kathi ...................................108Schilling, Frank ........................................126Schillinger, Christian .................................44Schlegel, Brigitte ......................................52Schlundt, Andreas ....................................40Schmeisser, Edelgard .............................112Schmieder, Peter .......................................50Schmidt, Antje ..........................................88Schmidt, Mathias ....................................126Schmikale, Bernhard ...............................104Schmoranzer, Jan ......................................76Schnuppe, Kristin ......................................68Schnurr, Matthias ......................................56Schröder, Leif ......................................16, 54Schülein, Ralf .....................................86, 126Schulz, Katharina .....................................126Schulz, Pascal ..........................................126Schumacher, Dominik .............................100Schumacher, Gabriele ............................126Schümann, Michael ................................117Schütz, Irene ..............................................76Schütze, Sebastian ....................................68Schwarzer, Dirk ........................................122Seedorf, Sabine ........................................52Seidler, Patrick ...........................................68Seiter, Florian ............................................36Seja, Patricia ..............................................68Selenko, Philipp ........................................58Seyffarth, Carola .....................................120Shahid, Shakeel.........................................36Siebertz, Kristina .....................................100Specker, Edgar ........................................120Spitzmaul, Guillermo ................................68

Linden, Arne ..............................................36Liokatis, Stamatios ....................................60Lisurek, Michael ........................................48Lo, Wen-Ting.............................................76Lojek, Eva ................................................126Lorenz, Dorothea ......................................90Ludwig, Carmen ........................................68

MMaglione, Marta .......................................76Majkut, Paul .............................................100Mallow, Kevin ..........................................120Marat, Andrea Lynn ..................................76Maritzen, Tanja ..........................................76Markovic, Stefan .......................................36Martos, Vera ......................................84, 100Mauks, Silvia ............................................126McLean, Martin .......................................112Meiners, Torsten .....................................112Menßen, Adriane ....................................126Messing, Claudia ....................................126Mevert, Jürgen ........................................126Michl, Dagmar.........................................104Miksche, Sandra ......................................112Motzny, Kathrin .........................................40Mühlbauer, Maria ......................................76Mühlberg, Michaela ...............................100Müller, Daniela ..........................................48Müller, Matthias ........................................48Münch, Jonas ............................................68Mussehl, Marco .......................................126

NNeuenschwander, Martin .......................120Newie, Inga ...............................................72Nguyen, Thi-Bich ......................................36Nickeleit, Irina .........................................112 Nieto, Olaia .............................................100Nieuwkoop, Andy .....................................36Nikolenko, Heike ....................................108Nischan, Nicole .......................................100Nitschke, Norma .......................................68

Spors, Marina ..........................................126Staat, Christian ..........................................72Stahlschmidt, Wiebke ..............................76Stauber, Tobias ..........................................68Stechmann, Bahne ..................................112Stephanowitz, Heike ...............................117Steuer, Andrea ........................................126Sticht, Jana ................................................40Stuhlmann, Till ..........................................68Stuiver, Marchel.........................................60Sydow, Karl ..............................................108

TTeichmann, Anke ......................................92Teschke, Till .............................................124Theillet, Francois-Xavier ...........................60Thomsen, Susanne ...................................76Thongwichian, Rossukon .........................60Tscheik, Christian ......................................72

UUllrich, Florian ...........................................68Uschner, Michael .....................................126

VVallée, Robert ..........................................100van Rossum, Barth ....................................36van Rossum, Marleen ...............................60Verzini, Silvia ..............................................60Vogelreiter, Gabriela ...............................108von Bock, Anyess ......................................68Voreck, Anja ..............................................36Voß, Felizia ................................................68Vukoja, Anela ............................................76

WWartenberg, Anne ....................................36Weidlich, Andrea ......................................68Weimer, Renate .......................................126Weinert, Stefanie ......................................68Wendt, Stephanie ...................................126Westendorf, Carolin .................................88

OOberheide, Karina ....................................68Oder, Andreas .........................................120Oestreich, Sylvia .....................................112Opitz, Robert.............................................48Oppmann, Birgit .....................................126Orozco, Ian ................................................68Orwick, Marcella .......................................36Oschkinat, Hartmut ..................................34Oßwald, Silke ..........................................126Otto, Christel ..........................................126Otto, Henning .........................................126Özdogan, Tugba .......................................80

PPál, Balazcs ................................................68Panzer, Holger .........................................126Pareja, Ruth ...............................................68Pechstein, Arndt .......................................76Pereplittchenko, Lioudmila ....................112Petschick, Heidi .......................................126Piontek, Anna ............................................44Piontek, Jörg .............................................72Piotukh, Kiril ........................................40, 48Planelis-Cases, Rosa .................................68Plested, Andrew J. R. ..........................12, 82Ponomarenko, Alexey ..............................78Posor, York .................................................76Protze, Jonas .......................................44, 72Prütz, Lisa.................................................126Puchkov, Dmytro .......................................76

RRäbel, Katrin ..............................................68Radetzki, Silke .........................................120Rehbein, Kristina .......................................36Reif, Bernd .................................................24Reimann, Oliver ......................................100Reinke, Stefan .........................................100Reiske, Simon ..........................................100Retel, Joren Sebastian ..............................36Ridelis Rivas, Ingrid ...................................88Ringler, Mario ............................................68Ringling, Martina ......................................92Röben, Marco ............................................52

Wieczorek, Marek .....................................40Wiesner, Burkhard.....................................90Wietstruk, Marcus .....................................84Wilkening, Ina .........................................100Winkler, Lars ..............................................72Wippich, Marc .........................................120Witte, Christopher ....................................56Wojtke, Susanne .......................................76Wolschke, Roy .........................................126

ZZampatis, Dimitris .....................................88Zapke, Janet ......................................36, 126Zillmann, Silke ...........................................68Zimmer, Dietmar .......................................68Zwanziger, Denise .....................................72

130 131

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2 F O R S C H U N G S B E R I C H T 2 0 1 1 / 2 0 1 2 C A M P U S B E R L I N B U C HC A M P U S B E R L I N B U C H

RESEARCH Leibniz-Institut für Molekulare Pharmakologie (FMP) C 81 Leibniz-Institut für Molekulare PharmakologieC 81.1 NMR IC 81.2 NMR 2 Max Delbrück Center for Molecular Medicine (MDC) C 27 Walter-Friedrich-House C 31 Max-Delbrück-House C 83 Max Delbrück Communications Center C 84 Hermann-von-Helmholtz-House A 10 Library B 63 Research services Shared Facilities by MDC and FMP C 84.1 Research servicesC 87 Timoféeff-Ressovsky-Haus CLINICAL RESEARCH

COMMON FACILITIES A 8 Gate House with Café Max A 9 Reception A 13 Life Science Learning Lab; Campus-Info-Center A 14 Cafeteria Guesthouses of the MDC B 54 Hans-Gummel-Guest House B 61 Kindergarten; Salvadore-Luria-Guest House COMPANIES

C A M P U S B E R L I N B U C H

132

R E S E A R C H R E P O R T 2 0 1 1 / 2 0 1 2I M P R I N T

I M P R I N T

Leibniz-Institut für Molekulare Pharmakologie (FMP) im Forschungsverbund Berlin e.V. Campus Berlin-Buch Robert-Rössle-Str. 10 13125 Berlin Germany

Phone + 49 30 947 93 - 104 Fax + 49 30 947 93 - 109 E-mail osswald@fmp-berlin.de

www.fmp-berlin.de

Research Report 2011/2012 Editorial Board Christian HackenbergerVolker Haucke Thomas JentschHartmut Oschkinat

Coordination Silke OßwaldHenning Otto

Author Feature Articles and Interview Birgit Herden

Editing Russ HodgeMartin McLeanSilke OßwaldHenning Otto

TranslationsClaudia Hecker, Mick Locke

Photography Silke Oßwald

Further Photography Maj Brit Jansen (p. 66, 70)

3-D IllustrationsMichael Lisurek Barth van Rossum

Scientific Figures SectionsMichael Lisurek (p. 95)Barth van Rossum (p. 31)Jan Schmoranzer (p. 63)

Design and Layout Apfel Zet, Berlin

Print NEUNPLUS1Verlag + Service GmbH, Berlin

Berlin, August 2013