ACTAUNIVERSITATIS
UPSALIENSISUPPSALA
2014
Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 1133
Revealing Secrets of SynapticProtein Interactions
A Biosensor based Strategy
CHRISTIAN SEEGER
ISSN 1651-6214ISBN 978-91-554-8916-8urn:nbn:se:uu:diva-220879
Dissertation presented at Uppsala University to be publicly examined in B42, Husargatan3, 751 23 Uppsala, Friday, 16 May 2014 at 13:15 for the degree of Doctor of Philosophy.The examination will be conducted in English. Faculty examiner: Professor Sarah Lummis(University of Cambridge).
AbstractSeeger, C. 2014. Revealing Secrets of Synaptic Protein Interactions. A Biosensorbased Strategy. Digital Comprehensive Summaries of Uppsala Dissertations from theFaculty of Science and Technology 1133. 73 pp. Uppsala: Acta Universitatis Upsaliensis.ISBN 978-91-554-8916-8.
Protein interactions are the basis of synaptic function, and studying these interactions ona molecular level is crucial for understanding basic brain function, as well as mechanismsunderlying neurological disorders. In this thesis, kinetic and mechanistic characterization ofsynaptic protein interactions was performed by using surface plasmon resonance biosensortechnology. Fragment library screening against the reverse transcriptase of HIV was included,as it served as an outlook for future drug discovery against ligand-gated ion channels.
The protein-protein interaction studies of postsynaptic Ca2+ -binding proteins revealedcaldendrin as a novel binding partner of AKAP79. Caldendrin and calmodulin bind and competeat similar binding sites but their interactions display different mechanisms and kinetics. Incontrast to calmodulin, caldendrin binds to AKAP79 both in the presence and absence of Ca2+
suggesting distinct in vivo functional properties of caldendrin and calmodulin.Homo-oligomeric β3 GABAA receptors, although not yet identified in vivo, are candidates for
a histamine-gated ion channel in the brain. To aid the identification of the receptor, 51histaminergic ligands were screened and a unique pharmacology was determined. A furtherrequirement for identifying β3 receptors in the brain, is the availability of specific high-affinity ligands. The developed biosensor assay displayed sufficient sensitivity and throughputfor screening for such ligands, as well as for being employed for fragment-based drug discovery.
AMPA receptors are excitatory ligand-gated ion channels, involved in synaptic plasticity,and modulated by auxiliary proteins. Previous results have indicated that Noelin1, a secretedglycoprotein, interacts with the AMPA receptor. By using biochemical methods, it wasshown that Noelin1 interacts directly with the receptor. The kinetics of the interactionwere estimated by biosensor analysis, thereby confirming the interaction and suggesting lownanomolar affinity. The results provide a basis for functional characterization of a novel AMPAreceptor protein interaction.
The results demonstrate how secrets of synaptic protein interactions and function wererevealed by using a molecular based approach. Improving the understanding of such interactionsis valuable for basic neuroscience. At the same time, the technical advancements that wereachieved to study interactions of ligand-gated ion channels by surface plasmon resonancetechnology, provide an important tool for discovery of novel therapeutics against these importantdrug targets.
Keywords: Surface plasmon resonance, biosensor, AMPA receptor, GABAA receptor, ligand-gated ion channel, A-kinase anchoring protein, caldendrin, calmodulin, HIV, fragment baseddrug discovery
Christian Seeger, Department of Chemistry - BMC, Biochemistry, Box 576, UppsalaUniversity, SE-75123 Uppsala, Sweden.
© Christian Seeger 2014
ISSN 1651-6214ISBN 978-91-554-8916-8urn:nbn:se:uu:diva-220879 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-220879)
Curro ergo sum
List of papers
This thesis is based on the following papers, which are referred to in the textby their Roman numerals.
I Xenia Gorny, Marina Mikhaylova, Christian Seeger, PashamParameshwar Reddy, Carsten Reissner, H. Bjorn Schott, U. HelenaDanielson, R. Michael Kreutz, and Constanze Seidenbecher.AKAP79/150 interacts with the neuronal calcium-binding protein
caldendrin. Journal of Neurochemistry, 122(4):714–726, 2012.
II Christian Seeger, Xenia Gorny, Pasham Parameshwar Reddy,Constanze Seidenbecher, and U. Helena Danielson. Kinetic and
mechanistic differences in the interactions between caldendrin and
calmodulin with AKAP79 suggest different roles in synaptic
function. Journal of Molecular Recognition, 25(10):495–503, 2012.
III Christian Seeger, Tony Christopeit, Karoline Fuchs, Katharina Grote,Werner Sieghart, and U. Helena Danielson. Histaminergic
pharmacology of homo-oligomeric β3 γ-aminobutyric acid type a
receptors characterized by surface plasmon resonance biosensor
technology. Biochemical Pharmacology, 84(3):341–351, 2012. 1
IV Nikhil Pandya, Christian Seeger, Renato Frischknecht, Sabine Spijker,Ka Wan Li, U. Helena Danielson, August B. Smit. Extracellular
matrix protein noelin1 interacts with the AMPA receptor.
manuscript.
V Matthis Geitmann, Malin Elinder, Christian Seeger, Peter Brandt, Iwande Esch, U. Helena Danielson. Identification of a novel scaffold for
allosteric inhibition of wild type and drug resistant HIV-1 reverse
transcriptase by fragment library screening. Journal of MedicinalChemistry, 54(3):699–708, 2011.
Reprints were made with permission from the publishers.
1Christian Seeger, Tony Christopeit, Karoline Fuchs, Katharina Grote, Werner Sieghart, andU. Helena Danielson. Corrigendum to “histaminergic pharmacology of homo-oligomeric β3γ-aminobutyric acid type a receptors characterized by surface plasmon resonance biosensortechnology”[biochem. pharmacol. 84 (2012) 341–351]. Biochemical Pharmacology, 2012.
Contents
1 Unexplored continents of the brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2 The chemical synapse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122.1 Postsynaptic density . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2.1.1 A-kinase anchoring protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132.1.2 Ca2+-binding proteins - calmodulin and caldendrin . . . . . . 14
2.2 Ligand-gated ion channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162.2.1 GABAA receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172.2.2 Ionotropic glutamate receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192.2.3 Ligand-gated ion channels as drug targets . . . . . . . . . . . . . . . . . . . . . 22
3 SPR biosensor technology for biomolecular interaction analysis . . . . . . . . . . 243.1 The detection unit of SPR biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243.2 Performance of SPR based interaction studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
3.2.1 Immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263.2.2 Interaction analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273.2.3 Biomolecular interaction mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . 283.2.4 Kinetic evaluation of biosensor data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
3.3 Fragment based drug discovery using SPR biosensortechnology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
3.4 SPR based interaction studies of membrane proteins . . . . . . . . . . . . . . . . . . 333.4.1 On-surface reconstitution of membrane proteins . . . . . . . . . . 333.4.2 Detergent-solubilized proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343.4.3 StaRs - Stabilized receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
4 Present Investigation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364.1 Identification of a novel postsynaptic protein-protein interaction
(Paper I) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364.1.1 Minimal binding site for caldendrin and calmodulin . . . . 374.1.2 Qualitative kinetic analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
4.2 Characterization of a novel postsynaptic protein-proteininteraction (Paper II) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394.2.1 Can biosensor data suggest protein function? . . . . . . . . . . . . . . . 39
4.3 Characterization of ligand-gated ion channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 414.4 Pharmacological evaluation of homo-oligomeric β3 GABAA
receptor (Paper III) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 424.4.1 Development of SPR biosensor assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
4.4.2 GABA binding to β3 GABAA receptors . . . . . . . . . . . . . . . . . . . . . . . 434.4.3 A candidate for a histamine-gated ion channel . . . . . . . . . . . . . 45
4.5 Identification and characterization of novel AMPA receptorprotein interaction (Paper IV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 474.5.1 SPR assay for protein interaction studies of AMPA
receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 484.5.2 Direct interaction of Noelin1-3 with GluA2 receptor . . . 49
4.6 Fragment screening for identification of novel ligands (PaperV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 514.6.1 Primary screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 524.6.2 Competition and inhibition screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 534.6.3 Quantification of affinity and inhibition . . . . . . . . . . . . . . . . . . . . . . . . . 534.6.4 Implications for fragment based drug discovery against
ligand-gated ion channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
5 Future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
6 Sammanfattning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
7 Zusammenfassung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
8 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Abbreviations
AKAP79 A-kinase anchoring protein 79AMPA α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acidCaBP Ca2+-binding proteinDDM n-dodecyl-β -D-maltopyranosideEF-hand helix-loop-helix motifFBDD fragment based drug discoveryGABA γ-aminobutyric acidGABAAR γ-aminobutyric acid type A receptorGPCR G-protein coupled receptorHisCl histamine-gated chloride channelHIV-1 RT human immunodeficiency virus type 1 reverse transcriptaseHTS high-throughput screeningIC50 half maximal inhibitory concentrationKD equilibrium dissociation constantLTD long-term depressionLTP long-term potentiationnAChR nicotinic acetylcholine receptorNMDA N-methyl-D-aspartateNMR nuclear magnetic resonanceNNRTI non-nucleoside reverse transcriptase inhibitorNRTI nucleoside reverse transcriptase inhibitorPKA cyclic adenosine monophosphate-dependent protein kinaseSAR structure activity relationshipSPR surface plasmon resonanceStaR stabilized receptor
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1. Unexplored continents of the brain
“El cerebro es un mundo que consta de numerosos continentes inexplorados ygrandes extensiones de territorio desconocido.”
This is a quote from the Spanish neuroscientist Santiago Ramon y Cajalthat he made about 100 years ago. In English it means “the brain is a worldconsisting of a number of unexplored continents and great stretches of un-known territory”. The understanding of the central nervous system, that Cajaldescribed as unexplored continents, has been greatly improved in the late 19th
century. This was due to the seminal work of Camillo Golgi and Cajal himself.Golgi developed a silver staining method (Golgi’s method) that allowed de-tailed characterization of the morphology of neurons. The method was furtherrefined by Cajal, who eventually could produce detailed histological stainingsof neurons [10]. One of Cajal’s famous drawings, a histological staining of aPurkinje neuron illustrating its tree-like structure, is shown Figure 1.1.
Figure 1.1. Drawing of a Purkinje neuron based on a histological staining by SantiagoRamon y Cajal [138].
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At the time of Cajal’s and Golgi’s research, there were two main theoriesabout the architecture of the central nervous system, namely the reticular the-ory and the neuron theory. According to the reticular theory, which was de-fended by Golgi, neurons are joined together in a continuous network. Cajal,on the other hand, claimed that the central nervous system is built up of in-dividual neurons. Eventually, the work of Cajal provided evidence for thistheory, leading to the accepted neuron doctrine [10].
The understanding of the brain has significantly improved since the era ofCajal and Golgi, driven by methodological advancements to study differentaspects of the brain. Among them are next-generation sequencing for high-throughput genomics, X-ray crystallography for protein structure determina-tion and high-resolution microscopy for detailed imaging studies. Neverthe-less, the brain is far from being fully understood and one major challenge isto improve the understanding and treatment of neurological disorders. Suchdisorders, especially Alzheimer’s disease and other forms of dementia, are es-timated to increase considerably until 2030, representing a great burden forhealth care services and individual suffering [96].
This thesis is an attempt to uncover parts of the unknown territory of ourbrain. The focus has been on aspects related to basic neuroscience, like thestudies of interactions between postsynaptic density proteins. Effort has beeninvested in the development of novel methods that could be applied in thediscovery of novel therapeutics for neurological disorders.
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2. The chemical synapse
Neurons are the basic units of our brain and their organization and connec-tions determine our behavior. It is estimated that the human brain containsapproximately 1×1011 neurons [3]. The regulation of such a large networkrequires special communication sites between the neurons. These sites are thesynapses, interfaces between the presynaptic bouton of one neuron and thepostsynaptic terminal of another neuron. The basic organization of a chemicalsynapse is illustrated in Figure 2.1. Large neuronal networks are formed bythe ability of a single neuron to form many synaptic contacts. A single neuroncan form approximately 1×104 contacts, while Purkinje neurons of the cere-bellum (Figure 1.1), can form up to 1×105 synaptic contacts [58].
Figure 2.1. Scheme and components of a chemical synapse.
Signal transmission in a chemical synapse is a process involving a largevariety of different proteins, small organic molecules and ions. A presynap-tic action potential leads to an increase of intracellular Ca2+ that triggers thefusion of synaptic vesicles with the presynaptic membrane at the active zone.Neurotransmitters are released into the synaptic cleft and diffuse to the postsy-naptic membrane where they bind to ligand-gated ion channels. The bindingopens the channels and leads to an ion flux, thereby changing the membrane
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potential at the postsynaptic terminal, and to the propagation of an action po-tential [58]. Synaptic function is regulated through both the postsynaptic den-sity and the extracellular matrix, protein dense networks involving a variety ofprotein-protein interactions.
The majority of synapses in the brain are chemical. These synapses are ableto perform more complex tasks than electrical synapses, which are mainlyinvolved in sending rapid and depolarizing signals. For example, chemicalsynapses are involved in mediating inhibition, excitation and amplification ofneuronal signals [58]. Amplification is an important feature of neuronal signaltransmission. The fusion of a synaptic vesicle with the presynaptic membrane,releases a large number of neurotransmitters that can open a similarly largenumber of ligand-gated ion channels.
Chemical synapses can be divided into two classes, namely excitatory andinhibitory synapses. Excitatory synapses use glutamate as their major neuro-transmitter and are referred to as glutamatergic synapses. Inhibitory synapses,use γ-aminobutyric acid (GABA) as their major neurotransmitter and are re-ferred to as GABAergic synapses [58]. Excitatory and inhibitory synapsesare not only distinguished by their major neurotransmitter, they also differ inmorphology. Excitatory synapses have an asymmetric morphology and aretermed Gray Type I synapses [58]. The postsynapse of Gray Type I synapsesis larger than the presynapse due to the pronounced postsynaptic density, aprotein-dense network, while Gray Type II synapses have a less pronouncedpostsynaptic density and appear symmetric [30].
2.1 Postsynaptic densityThe postsynapse, especially of excitatory Gray Type I synapses, contains adense protein network, known as the postsynaptic density, that consists of sev-eral hundred proteins [28]. The rearrangement of the postsynaptic density is adynamic process and basis for synaptic regulation and synaptic plasticity, likelong-term potentiation (LTP) and long-term depression (LTD) (Section 2.2.2).There are many different protein classes within the postsynaptic density, ofwhich scaffolding proteins, Ca2+-binding proteins (CaBPs) and membrane-bound receptors and channels will be described below [12] .
2.1.1 A-kinase anchoring proteinA-kinase anchoring protein 79 (AKAP79) is a human scaffolding protein thatwas identified as a component of the postsynaptic density [17]. Differentorthologs exist in several species that vary in their molecular weight (hu-man: AKAP79, bovine: AKAP75 [13], murine: AKAP150 [110]). AKAP79
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was originally named on the basis of its function to anchor cyclic adenosinemonophosphate-dependent protein kinase (PKA) [51] to specific intracellularsites, which is important for cyclic adenosine monophosphate signal transmis-sion. By positioning PKA in close proximity to its phosphorylation targets,AKAP79 increases the specificity of this broadly acting kinase [99, 92].
AKAP79 does not only anchor PKA, it also forms other protein complexes,like with the phosphatase calcineurin [26], protein kinase C [66] and withCaBPs, like calmodulin [36]. The discovery and characterization of an inter-action between AKAP79 and caldendrin, a further CaBP, is described PapersI and II. AKAP79 even plays a bifunctional role in its protein-protein interac-tions, for example by anchoring both PKA and calcineurin. PKA-dependentphosphorylation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid(AMPA) receptors during LTP increases the opening probability of the ionchannel and stabilizes the receptors upon internalization to the postsynapticmembrane. Calcineurin-dependent dephosphorylation of the same AMPA re-ceptor subunits during LTD leads to receptor internalization [36]. The bindingdomains of AKAP79 for the different interactions are illustrated in Figure 2.2.
Figure 2.2. Illustration of the binding domains of AKAP79. Protein kinase C, PKC,and calmodulin, CaM, bind to the A-domain (aa 31–52) [36]. CaM further binds to theB-domain, as well as caldendrin, CDD (Papers I and II). Calcineurin, CaN, and PKAbind to the C-terminus (aa 315–360 and aa 392–405). [17, 32]. LZ, leucine zipper.
2.1.2 Ca2+-binding proteins - calmodulin and caldendrinCa2+ signaling is highly universal and versatile, and controls processes likeproliferation and learning and memory [8]. By interaction with CaBPs, Ca2+
signals are associated with cellular and biochemical changes [22]. An impor-tant class of CaBPs is the helix-loop-helix motif (EF-hand) family of proteins,that includes calmodulin and caldendrin.
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Calmodulin is present in all eukaryotic cells and is the best studied CaBP. Itis a acidic protein with a molecular weight of approximately 17 kDa. Calmod-ulin contains four EF-hands, each of them able to bind a Ca2+ ion. Bindingof Ca2+ to apo calmodulin induces a conformational switch from a compactto an open form [70, 4]. This switch exposes a hydrophobic pocket in boththe N- and C-terminal domains of calmodulin which enables its interactionwith protein targets [74]. Generally, the compact form is regarded as inactiveand the open form as active. This is due to the fact that most proteins bind tocalmodulin in its open form, while only a few proteins, like neurogranin andneuromodulin bind preferably to apo calmodulin [137, 71]. The structures ofapo calmodulin and Ca2+/calmodulin are shown in Figure 2.3 illustrating theconformational switch that mainly occurs in the EF-hand domains.
Figure 2.3. Structures of Ca2+/calmodulin (left, 3cln.pdb), apo calmodulin (middle,1cfd.pdb) and Ca2+/caldendrin (right). Ca2+ ions are shown as red spheres.
Caldendrin is a neuron-specific protein that is particularly enriched in thepostsynaptic density of excitatory synapses [113]. Like calmodulin, calden-drin belongs to the EF-hand family of CaBPs. After its discovery, a subfamilyof CaBPs with five members (CaBP1-5) was identified, with caldendrin beinga splice variant of CaBP1 [45]. Sometimes, the term CaBP1 is used as a syn-onym for caldendrin. The structure of caldendrin (Figure 2.3) displays similar-ities with calmodulin but there are significant differences that make caldendrina unique CaBP. While the C-terminal domain of caldendrin displays stronghomology with calmodulin, the N-terminal domain is unique. Further differ-ences to calmodulin are related to the EF-hands. Two of the four EF-hands ofcalmodulin are conserved in caldendrin (EF-hand 3 and 4) while two EF-hands
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clearly differ (EF-hand 1 and 2) [113, 45, 75]. The equilibrium dissociationconstant (KD) of Ca2+ for binding to EF-hand 3 and 4 is in the low μM range,while it is considerably higher for EF-hand 1 (> 100μM) and not detectablefor EF-hand 2, which is referred to as cryptic [113, 134]. Another structuraldifference is that the central α-helix is four amino acids longer in caldendrincorresponding to one α-helical turn [45].
Compared to calmodulin, which has a large number of binding partners,only a few binding partners of caldendrin are known to date. In addition tointeracting with AKAP79, as described in Papers I and II, caldendrin interactswith the voltage-dependent L-type Ca2+ channel and modulates channel func-tion in a Ca2+-independent manner [125]. It binds to the C-terminal domainof the Ca2+ channel where it competes with calmodulin and CaBP1, but has adistinct regulatory effect. Caldendrin also interacts with the recently identifiedsynaptic protein, Jacob [33]. Jacob and caldendrin display a similar synap-tic distribution and their interaction is Ca2+-dependent. Caldendrin binds tothe nuclear localization signal of Jacob and prevents its trafficking from ex-tranuclear compartments to the nucleus, which in turn protects neurons fromN-methyl-D-aspartate (NMDA) receptor-dependent cell death [33].
2.2 Ligand-gated ion channelsThe output of a synaptic signal, whether it is inhibitory or excitatory, de-pends on the type of ligand-gated ion channel that is activated by the releasedneurotransmitters. Inhibitory signal transmission is mainly mediated by γ-aminobutyric acid type A receptors (GABAARs) and glycine receptors, bothpermeable for chloride ions. Excitatory signal transmission is mainly medi-ated via ionotropic glutamate receptors that are permeable for divalent cations.
Ligand-gated ion channels are anchored into the postsynaptic membrane asmultimeric assemblies, where they enable transformation of chemical signal-ing to electric signaling. When neurotransmitters are released, they diffusethrough the synaptic cleft and specifically bind to ligand-gated ion channels.This binding event triggers the rapid opening of the respective ligand-gated ionchannel, and enables ions to selectively flow through the channel. Dependingon the type of ions, synaptic signals are either excited or inhibited. The basalmembrane potential is around −70 mV. When an inhibitory neurotransmit-ter triggers the opening of an anion selective ligand-gated ion channel, theion flux causes the membrane potential to become more negative, describedas hyperpolarization. On the other hand, an excitatory neurotransmitter thatcauses the opening of a cation selective ligand-gated ion channel leads to de-polarization. The increase of the membrane potential over a threshold of ap-proximately −55 mV initiates an action potential that is propagated down an
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axon enabling neuron-to-neuron communication [58]. Hyperpolarization, onthe other hand prevents the propagation of an action potential.
2.2.1 GABAA receptorsGABA is the major inhibitory neurotransmitter in the central nervous systemand mediates its action mainly via GABAARs [95]. These receptors belong tothe superfamily of cysteine-loop ligand-gated ion channels that further com-prises the nicotinic acetylcholine receptor, serotonin type 3 receptors, glycinereceptor and a Zn2+-activated ion channel [95]. The ion pore of the recep-tors is formed by five membrane spanning subunits, each having a molecularweight of approximately 50 kDa [93]. The extracellular domain of each sub-unit contains the ligand-binding domain as well as the characteristic cysteine-loop [83]. Every subunit contains a transmembrane domain that is composedof four α-helices, forming intracellular loops.
A remarkable feature of GABAARs is their subunit heterogeneity. There are19 different subunits (α1–6, β1–3, γ1–3, δ , ε , θ , π , ρ1–3), representing thelargest set among mammalian ion channels [116]. Native receptors are mainlycomposed of two α , two β and one γ subunit [20, 127] (Figure 2.4). The
Figure 2.4. Schematic illustration of GABAA receptor structure and location of ligandbinding sites. Left: hetero-oligomeric receptor composed of α (blue), β (green) and γ(red) subunits. Right: homo-oligomeric receptor composed of β subunits.
subunit composition of GABAARs influences receptor function and pharma-cology. For instance, an αβ subunit interface is required to form the ligand-binding domain for GABA [118], while the allosteric modulation of GABAbinding by benzodiazepines requires an αγ interface [115] (Figure 2.4).
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Much of the structural information on GABAARs is derived from stud-ies of other ligand-gated ion channels as well as the soluble acetylcholine-binding protein. For example, the structure of the nicotinic acetylcholine re-ceptor (nAChR) from Torpedo marmorata, an electric ray, has been deter-mined at low resolution in 1993 and was further refined at 4 A resolution in2005 [129, 130]. Additionally, the acetylcholine-binding protein has servedas an important model system for cysteine-loop receptors. It is a soluble pen-tameric protein from the freshwater snail Lymnaea stagnalis, it is closely re-lated to the N-terminal domain of the α subunit of the nAChR and containsligand-binding sites for nicotine and acetylcholine [14]. The structure of theacetylcholine-binding protein has provided information about general featuresof the ligand-binding site of cysteine-loop ligand-gated ion channels [83]. Fur-thermore, structures of nAChR and the acetylcholine-binding protein havebeen used as templates for homology modeling of GABAARs. This has, forexample, resulted in a hypothesis of how diazepam binds within the benzodi-azepine site [104]. In recent years, bacterial cysteine-loop receptor homologshave emerged as model systems for eukaryotic ligand-gated ion channels astheir structures are less challenging to determine by X-ray crystallization. Forexample, the ligand-gated ion channel from Erwinia chrysanthemi [50] hasbeen co-crystallized with GABA and flurazepam and it was shown that GABAacts as an agonist, although with mM potency, and and that its binding is mod-ulated by flurazepam [120]. The ligand-gated ion channel from Gloeobacterviolaceus [11] has been co-crystallized with the general anesthetic propofol inan inter-subunit and intra-subunit binding site [91].
From benzodiazepine to GABAA receptor
In the late 1970’s, two studies revealed that a benzodiazepine receptor existsin the rat central nervous system [84, 121]. It was concluded that GABAergicneurons play an important role for benzodiazepine action as the density profileof diazepam binding sites corresponded well with the locations of GABAARs[84]. Both studies showed in radioligand binding assays that GABA did notdisplace radiolabeled [3H]diazepam, thereby excluding the possibility thatGABAARs contain high-affinity-binding sites for benzodiazepines.
Shortly after the discovery of the benzodiazepine receptor, it was shown thatGABA could stimulate benzodiazepine binding to benzodiazepine receptors[63]. It was concluded that the binding site for GABA and benzodiazepinesare functionally and structurally different, possibly due to GABAAR hetero-geneity, but that there might be a spatial interaction between the two receptors.The author’s conclusions pointed in the right direction, namely that benzodi-azepine receptors and GABAARs are part of the same protein complex. Aclear indication of this was provided by the copurification of [3H]muscimol(GABAAR agonist) and [3H]flunitrazepam binding sites [40]. A study bythe same authors had already demonstrated that GABAARs contain benzo-
18
diazepine binding sites and vice versa [39]. Based on these result, a modelof a GABAAR, containing different subunits for binding of GABA, benzodi-azepines and barbiturates, was proposed [94]. This model did not take the pen-tameric assembly of GABAARs into account, but it indicated that the receptorcomplex had a modular structure. About ten years later, the pentameric as-sembly of GABAARs was finally determined using electron microscopy [89].
Homo-oligomeric GABAA receptors
As mentioned above, native GABAARs are assembled as hetero-pentamers,mainly consisting of 2 α , 2 β and 1 γ subunit. The receptor studied in PaperIII was the homo-oligomeric β3 GABAAR, illustrated in Figure 2.4. Homo-oligomeric receptors of β1-3 receptor subunits are generally not regarded asGABAARs [94] because they lack an αβ subunit interface and are not gated byGABA [135, 18]. Furthermore, in the absence of an αγ interface, they are notmodulated by benzodiazepines, as illustrated in Figure 2.4. However, it hasbeen shown that even without the αβ interface, GABA can open the chloridechannel of homo-oligomeric β2 and β3 [82, 109] as well as β1 GABAARs[108], although with low potency.
Despite the controversies about possible GABA gating, homo-oligomericGABAARs are useful model systems for structural and pharmacological stud-ies [117]. The homo-oligomeric GABAARs formed of β2 and β3 subunitshave binding sites for general anesthetics, like propofol and etomidate, andthe barbiturate pentobarbital [18, 135, 117]. Although recombinant expressionis still challenging, expression of a single subunit avoids the problem of un-defined subunit composition and receptor stoichiometry. Ideally, biophysicalcharacterizations using surface plasmon resonance (SPR) biosensor technol-ogy should be performed with hetero-oligomeric GABAARs but attempts toexpress such receptors with a defined subunit composition and in sufficientlyhigh concentrations for such studies have so far not been successful.
2.2.2 Ionotropic glutamate receptorsGlutamate is the major excitatory neurotransmitter and also the most com-monly used neurotransmitter in the brain. Its effects are mediated by bothionotropic and metabotropic glutamate receptors. Ionotropic glutamate recep-tors are divided into three subtypes on the basis of selective agonists, namelyAMPA, NMDA and kainate [52]. Since the work in Paper IV specificallyconcerns AMPA receptors, these are the focus of the following sections.
Discovery of AMPA receptors
Upon their discovery in 1989, AMPA receptors were first assigned as kainatereceptors [53]. A subsequent study identified four glutamate receptors that
19
were assigned as AMPA receptors [64]. The sequences of the four receptorsshared approximately 60 % to 70 % similarity and the GluR-A sequence shared100 % similarity with the initially assigned kainate receptor GluR-K1. Al-though the four receptors interacted with kainate, the pharmacological charac-teristics suggested that they are AMPA receptors. The nomenclature of AMPAreceptors and other glutamate receptor subunits can sometimes be confusing.For instance, AMPA receptor subunits 1–4 are often referred to as GluR1–4 orGluRA1–4. According to the International Union of Basic and Clinical Phar-macology (www.iuphar-db.org) the AMPA receptor subunits should be namedGluA1–4 [27]. According to this nomenclature, the receptor that was studiedin Paper IV was a homo-tetrameric GluA2 AMPA receptor.
Structure of GluA2 AMPA receptor
AMPA receptors have a tetrameric assembly, and each subunit has an approx-imate molecular weight of 100 kDa [136, 106]. Most native AMPA receptorcomplexes in the brain are formed by pairs of either GluA1-GluA2 subunitsor GluA2-GluA3 subunits. Homo-tetrameric GluA1 AMPA receptors are onlypresent to a minor degree [133]. In 2009, the X-ray structure of a rat GluA2AMPA receptor was resolved at a resolution of 3.6 A [119]. Overall, the ar-chitecture of the AMPA receptor resembles the capital letter Y and containsthree major domains: a ligand-binding domain, a transmembrane domain andan N-terminal domain that plays an important role in the assembly, traffickingand localization of the receptor (Figure 2.5). The localization of the ligand-binding domain is an interesting feature of AMPA receptors. Compared toGABAARs and other cysteine-loop receptors, whose ligand-binding domain islocated between two subunits (Section 2.2.1), this domain is located within theindividual subunits [119], which might explain why homo-tetrameric AMPAreceptors are gated by glutamate. The transmembrane domain forms the ionchannel and is composed of three transmembrane helices.
AMPA receptors and synaptic plasticity
One of the fascinating abilities of the mammalian brain is the adaptation ofneuronal activity based on experience. This ability is often referred to assynaptic plasticity and regarded as the cellular basis of learning and mem-ory. The prototypical forms of synaptic plasticity are the processes of LTPand LTD, that are triggered by high-frequency and low-frequency stimulationof synaptic activity, respectively [25, 80]. Plasticity based changes in synap-tic activity can have long-lasting effects that last up to several days. LTP ismainly based on a large NMDA receptor-dependent increase of postsynap-tic Ca2+ concentration, that leads to activation of signaling cascades involv-ing protein kinases. The activation of these kinases leads to an increase ofAMPA receptor channel conductance and, more relevant, to an increased in-corporation of AMPA receptors into the postsynaptic membrane, resulting in
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Figure 2.5. Structure of homo-oligomeric GluA2 AMPA receptor (3kg2.pdb). Left:Side view of the receptor showing the three major domains of the receptor (ATD,amino-terminal domain; LBD, ligand-binding domain; TMD, transmembrane do-main). Right. Top view of the receptor.
increased synaptic activity (Figure 2.6). LTD, on the other hand, is the resultof a minor NMDA receptor-dependent increase of postsynaptic Ca2+ concen-tration that preferentially activates protein phosphatases. Activation of proteinphosphatases leads to the internalization of AMPA receptors, thereby reducingsynaptic activity (Figure 2.6) [25] .
Figure 2.6. Illustration of synaptic plasticity in excitatory synapses. Insertion ofAMPA receptors leads to LTP. Removal of AMPA receptors leads to LTD.
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2.2.3 Ligand-gated ion channels as drug targetsThe central role of ligand-gated ion channels makes them important drug tar-gets. While G-protein coupled receptors (GPCRs) are targeted by most drugs(≈ 25%), about 8 % of the currently approved drugs modulate ligand-gatedion channels [97]. For instance, GABAARs, that are the targets of benzodi-azepines, barbiturates as well as general anesthetics, have been the major tar-get for anxiolytic drug discovery [43]. From the discovery in the late 1950’sto the introduction of Librium in 1960 and Valium in 1963 (Figure 2.7), ben-zodiazepines have been a therapeutic success and are still among the mostprescribed drugs for treating insomnia and anxiety [107, 124]. The main ad-vantage of benzodiazepines over barbiturates is their inability to open the chlo-ride channel in the absence of bound GABA, thereby reducing side effects ofbarbiturate-induced channel opening. Current limitations of benzodiazepinetreatment are addiction and sedative effects when used as an anxiolytic. Thisis mainly due to the poor subtype-selectivity of benzodiazepines. These effectsare linked to different α GABAAR subunits, and there has been considerableprogress in the development of subtype-selective drugs.
Figure 2.7. Structures of Librium, Valium, RO4938581, an inverse agonist of α5GABAARs and Fycompa, a noncompetitive antagonist of AMPA receptors.
A ligand can be selective for different ion channel subtypes through differ-ent mechanisms. Either by binding to one subtype but not to another (bindingselectivity) or by binding to several subtypes but only having having an effectat one (functional selectivity) or both [107]. A GABAAR receptor ligand thatdisplays both binding and functional selectivity is RO4938581 (Figure 2.7),an inverse agonist at α5 subunits. RO4938581 has been shown to enhancecognition in animal trials and to reverse cognitive deficits in a mouse model ofDown’s Syndrome, without causing any side effects [5, 81].
AMPA receptors are key proteins in synaptic plasticity (Section 2.2.2).Therefore, they might appear to be the ideal drug targets for treatment of cog-nitive deficits. However, recent drug discovery efforts to target AMPA recep-tors for treatment of neurological diseases, like amyotrophic lateral sclerosis
22
and Alzheimer’s disease, have failed [19, 44]. Efforts to target AMPA recep-tors for the discovery of novel antiepileptic drugs have been more successful.Perampanel (Figure 2.7), a noncompetitive AMPA receptor antagonist, dis-played positive effects in clinical trials for the treatment of epilepsy and wasapproved in 2012 under the trade name Fycompa [46]. The discovery of per-ampanel was based on a combination of two high-throughput screening (HTS)assays, followed by structure activity relationship (SAR) studies [49].
Despite some promising results in drug discovery against ligand-gated ionchannels, as exemplified by Fycompa and benzodiazepines, there are still con-siderable challenges. Validation of novel drug targets is especially difficultdue to differences in the physiology of animal models and humans [57]. Asdescribed above, attempts to target AMPA receptors for treating amyotrophiclateral sclerosis and Alzheimer’s disease have failed in humans, despite initialpromising results in animal models [19]. Another challenge is that previousdrug discovery campaigns against ligand-gated ion channels, using in vivopharmacological approaches, were often performed without knowing the drugtarget [57]. Serendipity has thereby played a major role, exemplified by benzo-diazepines that were discovered by chance during a laboratory cleanup [124].The availability of high-throughput methods to screen large compound li-braries against ligand-gated ion channels has allowed to focus more on molec-ular based drug discovery and has demonstrated success in the discovery ofFycompa [46, 35]. Even with the availability of HTS methods for screen-ing against ligand-gated ion channels, like high throughput electrophysiology,a remaining challenge is the discovery of small-molecule leads with desir-able physicochemical properties [57]. This might be a result of the increasedmolecular weight and lipophilicity of HTS leads compared those discoveredbefore the HTS era [65].
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3. SPR biosensor technology for biomolecularinteraction analysis
SPR biosensors are biophysical instruments for kinetic and mechanistic char-acterization of biomolecular interactions in real-time. The first commercialSPR biosensor for the detection of biomolecular interactions was introducedin 1991 by Biacore (today part of GE Healthcare) [56] and the technology hasincreased in popularity since then. About 400 publications referred to the ap-plication of SPR biosensors in 1998 [86] and this number increased to morethan 1500 publications in 2009 [103]. The technology is useful in many re-search areas from basic research to drug discovery. For the work presented inthis thesis, SPR biosensor technology was employed for studying the follow-ing types of interactions:
• Protein - protein interactions (Papers I and II)• Membrane protein - small molecule interactions (Paper III)• Membrane protein - protein interactions (Paper IV)• Protein - fragment interactions (Paper V)
3.1 The detection unit of SPR biosensorsSurface plasmon resonance is an optical phenomenon that occurs at the inter-face of a noble metal and a dielectric, under conditions of total internal reflec-tion. Such conditions can be observed when light from a medium of higherrefractive index (n1) is reflected on the interface of a medium with lower re-fractive index (n2) at an angle of incidence (Θ) that is equal or greater than theangle of total internal reflection (Θc).
Surface plasmons are oscillating free electrons of a metal that behave likenonradiative evanescent field waves, which means that they are not losing en-ergy. The amplitude of this wave decays exponentially into the dielectric andthe metal [68]. By inciting light that is polarized perpendicular to the planeof incidence at the interface between the noble metal and the dielectric, anenergy transfer from the photons of the inciting light to surface plasmons canbe achieved. This energy transfer, which is called SPR, can be realized usingan experimental setup known as Kretschmann configuration that is illustratedin Figure 3.1 [68, 69]. A glass prism is coated with a thin layer of a noblemetal facing the dielectric. Light that is polarized perpendicular to the plane
24
of incidence, is totally reflected at the surface of the noble metal. Surfaceplasmon resonance occurs at a defined angle of incidence (Θresonance) that isdependent on the optical material properties and the wavelength of the incitinglight. When all parameters are kept constant, except the dielectric, Θresonancebecomes a function of its optical properties.
Figure 3.1. Illustration of the Kretschmann configuration for the excitation of surfaceplasmons in metals.
The detection unit of commercially available SPR biosensors, is based onthe Kretschmann configuration. A carboxymethylated dextran matrix is at-tached to the gold surface facing the flow system (sensor surface). Targetmolecules can be immobilized to the dextran matrix within the penetrationdepth of the evanescent field wave. The interaction of a ligand with an immo-bilized target changes the refractive index close to the sensor surface, which inturn changes Θresonance. These changes are monitored in real-time and plottedas a function of time in a sensorgram that contains the kinetic information ofan interaction, illustrated in Figure 3.2. The signal in a sensorgram is reportedin response units ([RU]) and for proteins, a shift of 1 RU corresponds to achange in surface density of approximately 1pg mm−2 [56, 122].
3.2 Performance of SPR based interaction studiesFor SPR based interaction studies, at least two binding partners are needed.The standard Biacore terminology refers to a ligand as the molecule that is im-mobilized to the sensor surface [56] and to an analyte as the injected moleculein solution [54]. In the following, the standard terminology will not be applied,instead the injected molecule is referred to as the ligand and the immobilizedmolecule is either generally referred to as protein or target molecule.
An SPR biosensor based experiment consists of a preparative step and ananalytical step. During the preparative step the target molecule is immobilized
25
Figure 3.2. Scheme of an SPR biosensor detection unit for biomolecular interaction.The detection unit is based on the Kretschmann configuration and traces the reductionof light intensity as a function of the SPR angle Θ. Plotting Θ as a function of timeresults in a sensorgram containing the information about the kinetics of an interaction.
onto the sensor surface. In the analytical step, ligand interactions with theimmobilized target molecule are monitored.
3.2.1 ImmobilizationTarget molecules can be immobilized to SPR biosensor surfaces by differenttechniques. This section will only cover the two methods that were employedfor the research leading to this thesis, namely immobilization by covalent im-mobilization using amine coupling, and non-covalent immobilization by affin-ity capture. A comprehensive summary of different immobilization techniquescan be found in the Sensor Surface Handbook from Biacore [48].
Amine coupling
Amine coupling is a covalent immobilization technique that utilizes primaryamine groups of the target molecule [55]. The approach consists of threemain steps, namely activation, target injection and deactivation. During thefirst step, carboxyl groups of the dextran matrix are activated by the injec-tion of a mixture of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide, leading to the formation of N-hydroxysuccinimide es-ters. Subsequently, the target molecule is injected over the activated surface.The target molecule is pre-concentrated at the sensor surface due to electro-
26
static interactions between its positively charged amine groups and negativelycharged carboxyl groups of the dextran matrix. Electrostatic pre-concentrationonly occurs when the pH of the sample buffer is lower than the isoelectricpoint of the target molecule. Through a nucleophilic attack, the activated estergroups are replaced by amine groups leading to the covalent immobilizationof the target molecule to the biosensor surface. The reaction of the primaryamine groups is not quantitative, thereby leaving a considerable amount of freereactive ester groups. These groups can potentially disturb subsequent inter-action analysis due to their electrostatic potential and need to be deactivated.This is accomplished by the injection of ethanolamine that transforms reactiveester groups into amide groups. The injection of ethanolamine will also re-move target molecules that have not formed a covalent bond, but were looselybound to the sensor surface, e.g. via electrostatic interactions. By compari-son of the baseline level before the activation and after the deactivation, theimmobilization level, i.e. the amount of immobilized protein, is determined.
Affinity capture
Affinity capture of target molecules is another common immobilization tech-nique. It involves covalent immobilization of a capture molecule, for examplean antibody or streptavidin, followed by the injection and capture of a targetmolecule. The capture molecule either recognizes a tag or a specific epitopeof the target molecule.
The affinity capture technique is especially useful when a defined orienta-tion of the target molecule on the sensor surface is desired. Amine couplingcan lead to heterogeneity of the immobilized target molecules and in somecases to binding artifacts [60]. In Papers III and IV, affinity capture was cho-sen for immobilization of ligand-gated ion channels to enable purification ofthe proteins directly on the surface. Furthermore, while amine coupling canbee too harsh for some proteins, affinity capture enables the protein to remainin a favorable environment during immobilization. For example, during aminecoupling, proteins are often immobilized in salt-free buffers at acidic pH, con-ditions that proteins with low stability do often not tolerate.
3.2.2 Interaction analysisOnce the sensor surface has been prepared, it is possible to use it to studyligand interactions. For this, concentration series of the ligands are typicallyprepared. These should ideally cover a range around the expected KD. Inaddition to the choice of the ligand concentrations, there is the choice betweendifferent injection modes that will be described in the following sections.
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Multi-cycle injections
The standard method for injecting ligands over an SPR biosensor surface is aserial injection, where every cycle is recorded separately. During the analysis,the sensorgrams from different cycles are then overlaid (Figure 3.4), both forvisualizing the different injections as well as for aligning the sensorgrams tocommon time points, e.g. the injection start. The kinetic constants are thendetermined by non-linear regression analysis. During multi-cycle injectionsthe surface is usually regenerated after every cycle to ensure that the bindingcharacteristics of the surface are as similar as possible for every ligand in-jection [61]. If regeneration is not possible, there is a risk that binding sitesare gradually blocked during the interaction analysis. Alternative methods arethen required because the standard algorithms for data analysis are not valid.
Single-cycle injections
The standard approach of injecting ligands using the above described multi-cycle injections is dependent on complete removal of bound ligand after ev-ery cycle. There are, however, interactions that cannot be regenerated andthat demand for alternative methods of injecting ligands over biosensor sur-faces. Such a solution that enabled kinetic analysis if tightly binding moleculeswas introduced in 2006, and is commonly referred to as single-cycle kinetics[61, 128]. Using this approach, ligands are sequentially injected in one cycle,without regenerating the surface after each injection. By using algorithms thataccount for the gradually decreasing binding capacity of the biosensor sur-face, it is possible to obtain reliable kinetic data. A positive consequence ofthis method is that the time required to perform a series of ligand injectionsdecreases. This type of injection was a great benefit for the analysis of theinteraction between caldendrin and AKAP79, described in Paper II.
3.2.3 Biomolecular interaction mechanismsBiomolecules can interact through a variety of different mechanisms and oneadvantage of using SPR biosensor technology is the ability to identify suchmechanisms. The three interaction mechanism, whose simulated sensorgramsare shown in Figure 3.3, were considered in the projects of the thesis.
1-step mechanism
The simplest biomolecular interaction follows a reversible 1-step mechanism(Scheme 3.1). The injected ligand A, and the immobilized target moleculeB, form a AB complex with kinetics determined by the association and dis-sociation rate constants k1 and k−1, the concentration of free A and B andthe complex AB. The forward interaction step can be described according toEquation 3.1 and the reverse step according to Equation 3.2.
d[AB]dt
= k1 ∗ [A]∗ [B] (3.1)
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0 300
0
1301-step
Time [s]
Respon
se[RU]
0 300
2-site
Time [s]
0 300
2-step
Time [s]
Figure 3.3. Simulated sensorgrams of three interaction mechanisms. Left: 1-step(Scheme 3.1). Middle: 2-site (Scheme 3.3). Right: 2-step (Scheme 3.3). Rate con-stants: k1 = 105 M−1 s−1, k−1 = 10−2 s−1, k2 = 104 M−1 s−1 (2-site), k2 = 104 s−1
(2-step), k−2 = 10−3 s−1. Concentrations: 16 nM to 250 nM.
A+Bk1−−⇀↽−−
k−1
AB
Scheme 3.1. Reversible 1-step mechanism for interaction between A and B.
−d[AB]dt
= k−1 ∗ [AB] (3.2)
The overall rate equation for forward and reverse steps is summarized in Equa-tion 3.3 [62].
d[AB]dt
= k1 ∗ [A]∗ [B]− k−1 ∗ [AB] (3.3)
Once the interaction reaches equilibrium ( d[AB]dt = 0), the rate constant of the
forward and reverse steps are equal. The ratio of the kinetic rate constants k1and k−1, at steady state, represents the KD of the interaction (Equation 3.4).
KD =k−1
k1=
[A]∗ [B][AB]
(3.4)
2-site mechanism
A reversible 2-site mechanism (heterogeneous ligand mechanism) describesthe interaction of a ligand with two independent binding sites (Scheme 3.2).Each binding event is described according to a 1-step mechanism (Equation3.3), resulting in two sets of kinetic rate constants (k1, k−1, k2, k−2) and a KDfor each interactions. Although interactions according to a 2-site mechanismexist, they may sometimes represent artifacts. For example, immobilizationof target molecules can sometimes lead to surface heterogeneity and an in-teraction that resembles a 2-site mechanism. The actual interaction, however,might follow a 1-step mechanism [60]. It is therefore important to use an
29
experimental design that allows confirmation or rejection of an interaction ofhigher complexity than a 1-step mechanism.
A+B1k1−−⇀↽−−
k−1
AB1
A+B2k2−−⇀↽−−
k−2
AB2
Scheme 3.2. 2-site mechanism of the interaction between A, B1 and B2.
2-step mechanism
When a ligand interacts with its target molecule, one of the binding partnersoften needs to undergo a conformational change to form a stable complex.Such a 2-step or induced fit mechanism is illustrated in Scheme 3.3. The
A+Bk1−−⇀↽−−
k−1
ABk2−−⇀↽−−
k−2
AB*
Scheme 3.3. 2-step mechanism of the interaction between A and B including a con-formational change of the AB complex.
ligand A forms a complex with the target molecule B and in a second step,the AB complex undergoes a conformational change to form a stable AB*conformation. The two steps of the interaction are described in Equation 3.5and Equation 3.6.
d[AB]dt
= (k1 ∗ [A]∗ [B]− k−1 ∗ [AB])− (k2 ∗ [AB]− k−2 ∗ [AB∗]) (3.5)
d[AB∗]dt
= k2 ∗ [AB]− k−2 ∗ [AB∗] (3.6)
The KD for a 2-step mechanism is different compared to that for a 1-step or 2-site mechanism, as both steps of the interaction need to be taken into account(Equation 3.7).
KD =k−1
k1∗ k−2
k−2 + k2(3.7)
Similarly, as for a 2-site mechanism, if experimental data are best describedby a 2-step mechanism, it is important to make sure an appropriate experi-mental design has been used and to perform control experiments to supportthe mechanistic hypothesis.
3.2.4 Kinetic evaluation of biosensor dataFor the majority of interactions that are monitored with SPR biosensors, akinetic analysis can be performed. For this, global non-linear regression anal-
30
ysis is used to determine kinetic rate constants and affinities. The most com-monly employed interaction models have been described above, being a 1-step(Scheme 3.1), 2-step (Scheme 3.3) and 2-site model (Scheme 3.2). Measuresto evaluate the quality of a fit are to analyze the residuals as well as visualinspection. Figure 3.4 illustrates the fit of a 1-step model to simulated data.
Time
Respon
se
Concentration
Figure 3.4. Kinetic and affinity evaluation of biomolecular interactions. Left: Sen-sorgrams are simulated for a 1-step interaction (black dashed). A 1-step interactionmodel is fitted to the sensorgrams using global-nonlinear regression analysis (gray).For affinity analysis, signals are extracted from the sensorgrams at steady-state (blackmarks). Right: Extracted report points plotted as a function of concentration and aLangmuir isotherm equation is fitted by non-linear regression analysis.
The data obtained from SPR biosensor analysis does not always allow de-termination of kinetic rate constants of an interaction. Usually, this occurswhen the interactions are very fast, and the data collection rate of the instru-ment is not sufficient to resolve the fast association and dissociation phases.Such fast interactions can be analyzed by a steady-state analysis in order to de-termine the KD. Instead of analyzing the complete binding curve, as during akinetic analysis, a steady-state analysis only employs report points, i.e. signalsextracted from the sensorgrams at specific time points. Report points are thenplotted as a function of the respective ligand concentration, as illustrated inFigure 3.4. The KD can subsequently be determined by non-linear regressionanalysis. Typically, an equation derived from the Langmuir isotherm equation,originally used to describe the adsorption of gas molecules to solid surfaces[73], is widely used for that purpose. It contains the concentration of the in-jected ligand ([L], [M]), the parameter for the KD ([M]), the theoretical signalwhen saturation is reached (Rmax, [RU]) and a parameter (m, [RU]) to correctfor minor baseline shifts after reference and blank subtraction (Equation 3.8).This equation has been applied for data analysis in Papers III and V.
R =Rmax ∗ [L]KD +[L]
+m (3.8)
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In addition, for detailed affinity analysis in Paper V, Equation 3.8 was furthermodified by including a parameter (u, [RU L mol−1]) that corrects for sec-ondary effects, like unspecific binding and concentration-dependent changesin bulk refractive index (Equation 3.9).
R =Rmax ∗ [L]KD +[L]
+u∗ [L]+m (3.9)
3.3 Fragment based drug discovery using SPRbiosensor technology
HTS has been successful in the discovery of novel drugs, but has often beenhampered by poor physicochemical properties of lead compounds [65]. As aconsequence, fragment based drug discovery (FBDD) has emerged as a strat-egy to identify potent and physicochemically suitable leads. The origin ofFBDD is usually attributed to a study for analysis of fragments by AbbottLaboratories who used nuclear magnetic resonance (NMR) in 1996 [114]. Inthis study, a library containing 1×104 compounds, with an average molecularweight of 213 Da, was screened by NMR. Fragments that bound to the targetwere optimized by SAR analysis. After a secondary screening, and anotherround of SAR analysis, two fragments that bound to two different sites withμM affinity were identified. After linking the fragments, a lead compoundwith low nM affinity was obtained.
Two main aspects are underlying FBDD. The first is the smaller size ofthe fragment library containing 1×103 to 1×104 compounds compared totypically 1×106 compounds in a HTS library [65, 114]. The reason for asmaller library is that the chemical space of fragments is smaller than thedrug-like chemical space. It has been estimated that the fragment-like spacecontains 14×106 to 44×106 molecules, while the drug-like space contains upto 1×1060 compounds [7]. Therefore, fragment libraries cover a larger chem-ical space than HTS libraries. The second aspect is the difference in molecularweight between fragments HTS compounds. Usually, fragments have a molec-ular weight of approximately 250 Da, while HTS compounds have a molecularweight of up to 600 Da [100]. Starting the hit-to-lead optimization with com-pounds of high molecular weight can lead to poor physicochemical propertiesdue to an increase in molecular weight and lipophilicity [100]. Furthermore,although HTS hits often have high affinity, their interactions with the bind-ing site are often suboptimal, as illustrated in Figure 3.5. Fragments, on theother hand, form favorable contacts within the binding pocket more easily, andthereby interact with the target protein more efficiently (Figure 3.5). Such fa-vorable interactions are referred to as ligand efficiency, a metric that relates the
32
binding free energy of a ligand to the number of non-hydrogen atoms [7, 72].
Figure 3.5. Illustration of HTS hit (left) and FBDD hit (right) interactions with a targetprotein. HTS hits often show lower ligand efficiency in target binding than fragmenthits.
Among the methods employed for FBDD, SPR biosensor technology hasemerged as an important option. The first fragment screening using SPR tech-nology was performed against the matrix metalloproteinase 12 [90] and thepopularity of FBDD by SPR has increased since then. In Paper V, a frag-ment library was screened against the human immunodeficiency virus type 1reverse transcriptase (HIV-1 RT). A hit that displayed a favorable resistanceprofile was identified. Further SPR based fragment screenings with relevancefor this thesis are those that were recently performed against both native andstabilized GPCRs (more in Section 3.4.3), like the β2 [2] and β1 [24] adrener-gic receptors. Also, an SPR assay has been developed for fragment screeningagainst the acetylcholine-binding protein [102] and this study together withthe initial SPR based characterization of the binding protein [41] led to thestudies of the homo-oligomeric β3 GABAAR as described in Paper III.
3.4 SPR based interaction studies of membrane proteinsStudying membrane proteins with biophysical methods, like SPR biosensors,is a great challenge. The method requires relatively pure proteins on the sensorsurface and these proteins need to be stable during the timecourse of the mea-surements. Different approaches are used to immobilize membrane proteinson biosensor surfaces, which will be described in the following sections.
3.4.1 On-surface reconstitution of membrane proteinsA method for functional immobilization of the GPCR rhodopsin was intro-duced as on-surface reconstitution [59]. The approach made use the L1 sensorchip [29], whose dextran matrix is derivatized with alkyl chains in order to an-chor lipophilic groups. During on-surface reconstitution, detergent solubilizedand purified rhodopsin was covalently immobilized onto sensor surfaces using
33
amine coupling [55]. Subsequently, rhodopsin was injected in micelles con-sisting of lipids and detergent. As the lipids adhered to the alkyl groups on thesensor surface, the detergent was washed away. The elution of the detergentcaused the lipids on the surface to form a lipid bilayer, thereby reconstitutingrhodopsin in a membrane environment. By using on-surface reconstitution,rhodopsin was functionally immobilized on the sensor surface [59].
The introduction of on-surface reconstitution offered several advantages forSPR based analysis of membrane protein interactions. The approach is rela-tively easy, allows immobilization of high protein densities on sensor surfacesand employs standard immobilization techniques [62]. A disadvantage of thisapproach is that it requires pure protein because during amine coupling, allproteins of a crude sample carrying primary amine groups could otherwise beimmobilized. The approach has been further developed by replacing aminecoupling with affinity capture for immobilization of two GPCRs (CXCR4,CCR5) [123]. The GPCRs from cell lysates were detergent solubilized andinjected over sensor surfaces that contained immobilized antibodies. Afterthe affinity capture, mixed micelles were injected to reconstitute the GPCRs.CXCR4 was functionally immobilized, shown by binding of a conformation-dependent antibody and a endogenous ligand. Compared to the original studyintroducing on-surface reconstitution, immobilization of an anti-CXCR4 anti-body allowed crude cell lysates to be injected and the receptors to be purifiedon the sensor surface. During affinity capture, the membrane proteins are notexposed 1 M ethanolamine, commonly used during amine coupling, therebyavoiding a potential loss of structural stability. Recently, on-surface reconsti-tution was successfully applied to the studies of interactions of inhibitors withfull length β -secretase [23].
3.4.2 Detergent-solubilized proteinsA simplified approach to immobilize membrane proteins compared to on-surface reconstitution was demonstrated by using the same chemokine recep-tors CXCR4 and CCR5 as described above. Instead of reconstituting the re-ceptors in a lipid environment, they were kept in a detergent-solubilized stateon the sensor surface [88]. Apart from the reconstitution step, the assay setupis similar to the modified on-surface reconstitution because the membrane pro-tein is immobilized on the sensor surface via affinity capture [123]. Althoughmembrane proteins are usually more stable in a membrane environment, thestudied GPCRs were sufficiently stabilized throughout the interaction analy-sis. The possibility of using detergent-solubilized GPCRs was demonstratedby screening 200 compounds against CXCR4 and CCR5 [87].
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Recently, novel antagonists were discovered by SPR based fragment screen-ing against the wild-type β2 adrenergic receptor using this method [2]. Thestudy clearly demonstrates that interaction analysis of detergent-solubilizedGPCRs can be useful also for other targets than the chemokine receptors de-scribed above. In Papers III and IV, the homo-oligomeric β3 GABAAR andGluA2 AMPA receptor were immobilized on SPR biosensor surfaces usingsimilar approaches.
3.4.3 StaRs - Stabilized receptorsOne of the main challenges when studying membrane proteins with biophysi-cal methods, is their poor stability outside their native membrane environment.To overcome this challenge, other strategies, in addition to finding suitable sol-ubilization conditions, have been pursued. An approach for biophysical char-acterization of stabilized GPCRs, named stabilized receptors (StaRs), was in-troduced in 2010 [105]. It involved mutagenesis of two GPCRs, the adenosineA2A and the muscarinic M1 receptor, until stable conformations were identi-fied. The resulting StaRs were locked in either an agonist or an antagonistconformation, excluding the possibility to study agonist and antagonist inter-actions simultaneously.
Biophysical characterization of StaRs has been facilitated by the ability toexpress the receptors in mg amounts. SPR based fragment screening againstβ1 adrenergic receptors, followed by SAR analysis, led to the identificationof novel high-affinity ligands. Furthermore, the structure of the stabilizedβ1 adrenergic receptor was determined by X-ray crystallization [24]. Alsoother, more protein consuming methods than SPR biosensor technology havebeen successfully employed for fragment-screening against StaRs. Target im-mobilized NMR screening against the stabilized adenosine A2A receptor hasbeen performed [21]. Compared to NMR in solution, which requires largemg amounts of protein, immobilizing the target on a solid support decreasesamount of protein to a few mg [131].
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4. Present Investigation
The function of our brain is to a large extent defined by synaptic protein in-teractions. Revealing the secrets of synaptic protein interactions, as the titleof this thesis suggests, therefore adds valuable information for neuroscienceon a molecular level. The studies included in this thesis represent differentperspectives and aspects of synaptic protein interactions. In Papers I, II andIV, the main focus was on confirming and characterizing novel synaptic pro-tein interactions. The real-time data obtained using SPR biosensor technologywas valuable for detailed kinetic and mechanistic analysis, that goes beyondrather simple “yes or no” answers obtained with more conventional methods.In Paper III, the pharmacology of a yet unidentified variant of a ligand-gatedion channel was defined. At the same time, a methodological advancementwas achieved by developing a biosensor assay for direct interaction analysisof ligand-gated ion channels, as well as for screening of small molecule li-braries for discovery of novel ligands. Paper V uses a non-synaptic protein asa model system to illustrate how FBDD against ligand-gated ion channels maybe performed in the future.
4.1 Identification of a novel postsynaptic protein-proteininteraction (Paper I)
AKAP79 is a scaffolding protein in the postsynaptic density of excitatorysynapses. It is involved in the regulation of synaptic transmission. It coordi-nates the phosphorylation of receptors by anchoring kinases, especially PKA,near their substrates [17]. AKAP79 interacts with a variety of synaptic pro-teins. Among them is calmodulin, a prototypical CaBP, that has been shownto regulate the activity of protein kinase C by competing with the kinase forbinding at the A-domain of AKAP79 [36]. In addition, caldendrin was iden-tified in this study as a novel AKAP79 interacting CaBP. Interaction of bothcaldendrin and calmodulin with AKAP79 was demonstrated by immunopre-cipitation in rat brain extracts, in vitro pull-down assays and by SPR basedbiosensor analysis, as described below. Furthermore, caldendrin was shownto co-localize with AKAP150, the murine ortholog of AKAP79, in primaryhippocampal neurons.
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4.1.1 Minimal binding site for caldendrin and calmodulinAKAP79 contains several domains with binding sites for a variety of synap-tic proteins (Section 2.1.1). The interactions between caldendrin and differentAKAP79 constructs were studied using pull-down assays and it was shownthat the N-terminal domain of AKAP79 harbored the binding site for calden-drin, more specifically, within the B1/2 domain (aa 61–97) (Figure 4.1). Fur-thermore, these experiments demonstrated that calmodulin binds to two bind-ings sites at AKAP79, one in the A-domain [36] and another in the B-domain(B2 domain, aa 75–97). Caldendrin and calmodulin therefore interact at over-lapping binding sites within the B-domain of AKAP79 but caldendrin requiresa larger binding site that extends more into the N-terminal domain (Figure 4.1).
Figure 4.1. Illustration of the binding domains of caldendrin and calmodulin withinthe B-domain of AKAP79. Caldendrin binds at the B1/2 domain (aa 61–97) andcalmodulin binds at the B2 domain (aa 75–97).
4.1.2 Qualitative kinetic analysisThe information that is obtained from pull-down assays is qualitative, showingwhether an interaction occurs or not. The use of SPR biosensor technology isoften motivated by the possibility to obtain some kinetic rate constants of theinteraction of interest. What is often overlooked is that the technology canalso provide mechanistic and kinetic information on a qualitative level.
For the SPR based experiments that are described here and in Paper II, atruncated construct of AKAP79 forming the B1/2 domain was used. It will bereferred to as AKAP79-B. The interactions of caldendrin and calmodulin withAKAP79-B were demonstrated by biosensor experiments that revealed thatthey differ qualitatively (Figure 4.2). The detailed quantitative kinetic analy-sis is further described in Paper II.
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0 120
0
1,300
Calmodulin
Caldendrin
+Ca2+
Time [s]
Respon
se[RU]
Figure 4.2. Sensorgrams of caldendrin (solid) and calmodulin (dashed) interactingwith AKAP79-B at concentrations of 250 nM, in the presence of 2 mM Ca2+.
Many characteristics of the interactions of caldendrin and calmodulin canbe inferred by visual inspection of the sensorgrams. Most important, the in-teractions differ clearly. Calmodulin reaches a steady-state within the timeof the injection and dissociates rapidly during the dissociation phase, indi-cating both relatively fast association and dissociation rates. In contrast, theinteraction of caldendrin is characterized by slower and biphasic associationand dissociation phases. Furthermore, before a new cycle was started, non-dissociated calmodulin could be completely released from its complex withAKAP79-B by injecting a Ca2+-free buffer. The same regeneration conditionscould not release caldendrin completely from its complex with AKAP79-B.This confirms the results from pull-down analysis (Paper I) that calmodulinand caldendrin show different Ca2+ dependencies with respect to their interac-tions with AKAP79-B.
So why was a qualitative analysis of the AKAP79-B interactions performedin Paper I, when a detailed quantitative kinetic analysis was performed for thesame interactions in Paper II? The answer is that the scientific question in Pa-per I did not require a detailed kinetic analysis. It was enough to show thatcaldendrin and AKAP79-B interact directly. Describing the interaction qual-itatively, by applying a method complementary to those employed by others,like immunoprecipitation and pull-down assays, was sufficient.
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4.2 Characterization of a novel postsynapticprotein-protein interaction (Paper II)
The results in Paper I showed, on a qualitative level, that the interactions ofcaldendrin and calmodulin are clearly different. Not only the kinetics differed,but also their Ca2+ dependencies. The aim of Paper II was therefore to providea better understanding of the underlying kinetic and mechanistic details of theinteractions of caldendrin and calmodulin with AKAP79-B.
4.2.1 Can biosensor data suggest protein function?The detected signal in SPR based interaction analysis is generally based ona binding event that changes the refractive index close to the sensor surface,and that changes the angle at which SPR occurs (Section 3.1). Although astructurally intact target protein can be assumed to be functional, it does notnecessarily mean its interaction with another molecule has any implication forits function. Nevertheless, it is still tempting to make assumptions about thefunctional consequences of the studied protein-protein interactions.
The qualitative analysis in Paper I showed that calmodulin associates anddissociates faster than caldendrin, which was confirmed by the quantitativeanalysis in Paper II. The interaction could be sufficiently described by fittinga reversible 1-step model to the experimental data (Scheme 3.1), as shown inFigure 4.3. The association rate of calmodulin, describing the initial complex
0 120 180
0
800
250 nM
125 nM
63 nM
31 nM
16 nM
8 nM
+Ca2+
Time [s]
Respon
se[RU]
Figure 4.3. Sensorgrams from the interaction between calmodulin and AKAP79-Bin the presence of 2 mM Ca2+. A 1-step model (Scheme 3.1) (gray) was fitted to theexperimental data (black).
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formation, was two orders of magnitude faster than of caldendrin, suggestingmore efficient calmodulin-AKAP79 complex formation in the synapse at μMCa2+ concentrations (Table 4.1). This was further supported by competitionanalysis in Papers I and II that showed that calmodulin outcompetes calden-drin for binding to AKAP79-B. Under high Ca2+ conditions it therefore seemsunlikely that caldendrin would have a significant regulatory function via bind-ing to AKAP79.
Table 4.1. Kinetic rate constants and affinities for the interactions of caldendrin andcalmodulin with AKAP79-B in the presence of 2 mM Ca2+.
Protein k1 [s−1 M−1] k−1 [s−1] k2 [s−1] k−2 [s−1] KD [nM]
Caldendrin 9×103 4×10−3 3×10−3 2×10−4 20Calmodulin 2×106 5×10−2 30
The SPR based interaction analysis in Paper I showed that caldendrin inter-acted differently with AKAP79-B than calmodulin. Although the experimen-tal data in Paper II did not give conclusive evidence for the proposed 2-stepmodel (Scheme 3.3) that was used for data analysis, it seems likely that aninduced-fit mechanism is underlying the interaction between caldendrin andAKAP79-B (Figure 4.4). It has been mentioned in Section 2.1 that the centralα helix of caldendrin is 4 amino acids longer than in calmodulin [45]. Thiselongated helix provides increased structural flexibility for caldendrin and maybe the underlying reason for its interaction according to an induced-fit mecha-nism to the extended B1/2 domain of AKAP79. This hypothesis is supportedby biophysical studies of caldendrin and the voltage-gated ion channel 1.2that show that caldendrin needs to undergo a conformational change to enablebinding of the N-terminal and C-terminal domain [37].
Caldendrin interacted with AKAP79-B both in the presence and absenceof Ca2+ although with different kinetic rate constants (Figure 4.4). The dif-ferences regarding the EF-hands of caldendrin and calmodulin might be thereason for this behavior. Upon discovery of caldendrin, it was hypothesizedthat two of the EF-hands might have lost their ability to bind Ca2+ [113]. Thishypothesis was later partly confirmed by biophysical studies of Ca2+ bindingto CaBP1, i.e. caldendrin, that showed that EF-hand 1 shows weak affinityfor Ca2+ binding (> 100μM) and that no binding of Ca2+ could be detectedat EF-hand 2 [134]. Inactivation of EF-hands has been suggested to be anevolutionary mechanism to increase the diversity of CaBP function in Ca2+
signaling [45]. Enabling caldendrin to interact with AKAP79 under low Ca2+
concentrations, where it would not be outcompeted by calmodulin, would leadto different regulatory function in vivo.
40
0 1,8000
400
250 nM
125 nM
63 nM
31 nM16 nM
+Ca2+Respon
se[RU]
0 1,800
0
130
500 nM
250 nM
125 nM
63 nM
-Ca2+
Time [s]
Respon
se[RU]
Figure 4.4. Sensorgrams of caldendrin interacting with AKAP79-B in the presence(top) and absence (bottom) of 2 mM Ca2+. The experimental data (black) is fitted witha 2-step model (Scheme 3.3, gray).
Another feature of the interaction between caldendrin and AKAP79 is thatonce the protein complex is formed, the slow dissociation of caldendrin (Ta-ble 4.1) would keep the complex tightly associated even when Ca2+ drops tonM concentrations. Thereby, transient effects in synaptic signaling by risingand lowering of intracellular Ca2+ concentrations could not be mediated by atight association of caldendrin to AKAP79. Instead, caldendrin would medi-ate a long-lasting effect once bound to AKAP79. However, there might be amechanism to release bound caldendrin from AKAP79 by binding of an otherpostsynaptic protein to either of the proteins.
4.3 Characterization of ligand-gated ion channelsSmall molecule-ligand-gated ion channel interaction are commonly studiedusing radioligand binding assays and electrophysiological methods, such aspatch- or voltage-clamp techniques. For studies of interactions between pro-teins and ligand-gated ion channels, immunoprecipitation and pull-down as-says are often employed. One of the major advantages of these methods is thatthey allow the study of the ligand-gated ion channels in an almost native en-vironment, such as cells or membrane preparations, thereby ensuring receptorstability. However, for structure based drug design, biophysical methods are
41
more suitable, primarily NMR and X-ray crystallography that provide struc-tural information about the binding of a lead or drug. But for biophysicalstudies of ligand-gated ion channels, the stabilizing membrane typically needsto be removed and replaced by detergent. Solubilization is one of the majorchallenges in the application of biophysical methods for characterization ofligand-gated ion channels. Furthermore, both NMR and X-ray crystallogra-phy require protein amounts in the mg range. An alternative for biophysicalanalysis is SPR biosensor technology that was employed in this thesis to studyhomo-oligomeric GABAARs and AMPA receptors. While the ligand-gatedion channels still needed to be solubilized, the requirement with respect to theamount of protein is considerably lower and lies in the low μg range.
4.4 Pharmacological evaluation of homo-oligomeric β3GABAA receptor (Paper III)
4.4.1 Development of SPR biosensor assayBased on the success of applying on-surface reconstitution for GPCRs andβ -secretase 1 (Section 3.4.1) [59, 123, 23], a similar strategy was pursued forimmobilizing β3 GABAARs. However, this strategy did not allow interactionswith the receptor to be monitored. Therefore, an alternative strategy was em-ployed, keeping β3 GABAARs in a detergent-solubilized state throughout theinteraction analysis. This strategy has been successfully applied for SPR basedinteraction studies of GPCRs, like chemokine receptors [88] and recently theβ2 adrenergic receptor [2]. Although the assay setup becomes simpler com-pared to on-surface reconstitution, the major challenge, to identify suitablesolubilization conditions, remains. A rational approach has previously beenemployed to screen for solubilization conditions for chemokine receptors in ascreening-like manner [88]. As a consequence of the inability to regeneratethe antibody surface after affinity capture of the β3 GABAAR, such a deter-gent screening was not performed. Suitable solubilization conditions for theβ3 GABAARs were instead identified by trial and error. The receptor wassolubilized in a mixture of detergents and lipids. During the biosensor basedinteraction analysis, the receptor was kept solubilized in the presence of thenon-ionic detergent n-dodecyl-β -D-maltopyranoside (DDM). In a previousstudy, employing similar solubilization and buffer conditions, it was demon-strated that the stability of a hetero-oligomeric GABAAR was higher in a sol-ubilized state than in a reconstituted environment [34]. These findings supportthat the solubilization conditions employed in Paper III were suitable for in-teraction studies of the β3 GABAAR.
Once the solubilization conditions were identified, β3 receptors were im-mobilized on SPR biosensor surfaces via affinity capture using polyhistidine
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antibodies, as described in Section 3.2.1 (Figure 4.5). To prepare suitablereference surfaces, solubilized cell lysates from non-transfected cells were in-jected over a second, but similarly prepared antibody surface. By comparingthe signal levels on the β3 sensor surfaces with the control surfaces it was con-cluded that β3 receptors were specifically immobilized and the SPR biosensorassay was ready to be used for interaction studies.
Figure 4.5. SPR sensor surfaces for interaction studies of ligand-gated ion channels,LGIC. Antibodies, Ab, against the ligand-gated ion channel are covalently immobi-lized to empty biosensor surfaces. Detergent solubilized ion channels are immobilizedvia affinity capture, forming receptor surfaces. Control surfaces are formed from sol-ubilized membranes of non-transfected cells.
4.4.2 GABA binding to β3 GABAA receptorsControversial results regarding GABA gating of homo-oligomeric β receptorswere described in Section 2.2.1. GABA has been shown to have low potencyat β3 receptors [109, 108], while other studies mention that this type of recep-tor is insensitive to the neurotransmitter [135, 18]. In the present SPR basedinteraction analysis, a possible interaction of GABA was studied, but no bind-ing of GABA was detected at concentrations of up to 100 mM. The differentresults regarding the gating of homo-oligomeric GABAARs by GABA mightbe a result of different expression systems used in the previous studies [82].Different expression systems can lead to minor changes in the sequence ofthe receptors as well as to different posttranslational modifications. For ex-
43
ample, while the neurosteroid alphaxalone was shown to interact with rat β3GABAARs in a radioligand binding assay [117], mouse β3 receptors were in-sensitive to this ligand [135]. Alphaxalone was analyzed in Paper III but theresults could not be rationally interpreted.
The binding of general anesthetics, like propofol, etomidate and etazolate,as well as the binding of ligands to the peripheral benzodiazepine receptor,PK-11195 and Ro5-4864, to β3 receptors could be confirmed (Table 4.2).Although the KDs determined in the SPR biosensor study were about one or-der of magnitude higher than determined half maximal inhibitory concentra-tions (IC50s) in radioligand binding studies [117], they can be regarded to bein reasonable agreement. Binding of pentobarbital, a barbiturate, and alphax-alone, as mentioned above, could not be reliably detected in the current study.Binding of etomidate and propofol suggests that binding sites in the trans-membrane domain are structurally intact after detergent solubilization. Propo-fol has been co-crystallized with a ligand-gated ion channel from Gloeobacterviolaceus, and it was hypothesized that the binding site in the transmembranedomain might be the general site for general anesthetics in ligand-gated ionchannels [91]. The binding site of the general anesthetic etomidate was identi-fied in the transmembrane domain at the αβ interface by photoaffinity labeling[76]. Due to the lack of an α subunit, the results suggest that there is a bindingsite for etomidate either at the ββ interface or within a β subunit.
In conclusion, the homo-oligomeric β3 GABAAR that was studied in PaperIII does not interact with GABA. But the β3 receptors interacted with knownGABAergic ligands similarly as in HEK cell membranes, despite being stud-ied in a detergent-solubilized state.
Table 4.2. Comparison of affinities (KD) and potencies (IC50) of GABAergic ligandsat β3 receptors obtained by SPR biosensor and radioligand binding analysis [117].
Ligand KD [μM] IC50 [μM]
Etomidate 38 2Propofol 42 20Ro5-4864 69 9Etazolate 79 4Pentobarbital nd 20Alphaxalone nd 12nd, not detected
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4.4.3 A candidate for a histamine-gated ion channelThe β3 GABAAR does not only carry binding sites for general anesthetics,but also for histamine, acting as an agonist [109]. Histamine has also beenshown to potentiate GABA evoked currents of hetero-oligomeric GABAARs[109], especially those containing α4 subunits [9]. It was also hypothesizedthat cimetidine-like drugs produce seizures via GABAARs [16]. Although in-hibitory effects of histamine type 2 receptor antagonists, such as famotidineand tiotidine, were demonstrated at hetero-oligomeric GABAARs, the poten-cies were too low to cause seizures.
There is pharmacological and physiological evidence for the existence ofyet unidentified histamine-gated chloride channels (HisCls) in the mammalianbrain [139, 47]. In invertebrates two HisCls (HisCl α1 and HisCl α2) havebeen identified that are homologous with subunits of glycine receptors andGABAARs, including β3 GABAAR subunits [140, 42, 38]. Recently, his-tamine has been shown to act as an inverse agonist of hetero-oligomeric α1βglycine receptors [67].
Although it is tempting to believe that β3 GABAARs are the yet unidenti-fied HisCl in the mammalian brain, there are objections against this hypothe-sis. So far, homo-oligomeric GABAARs have not been identified in vivo and itis not clear whether they exist at all. Furthermore, mammalian HisCls in neu-rons, as described previously [47, 139] are blocked by cimetidine at low μMconcentrations. Cimetidine was shown to neither bind (Paper III) nor have aneffect on β3 GABAARs in electrophysiological studies [109]. However, evenif the homo-oligomer β3 GABAAR is not the missing HisCl, the β3 subunitcould play an important role in a potential hetero-oligomeric HisCl.
Histaminergic pharmacology of β3 GABAA receptors
One of the main goals in the research leading to Paper III, was to employ SPRbiosensor technology to expand the known histaminergic pharmacology of β3GABAARs. For this, a library of known histaminergic ligands to histaminetype 1–4 receptors was screened. Identifying a specific pharmacology that hasalready been indicated [109], and additionally discovering high-affinity lig-ands, could lead to the in vivo identification of β3 GABAARs.
The interaction of histamine, the endogenous ligand to all four histaminereceptors, was detected with signal levels close to the detection limit (Fig-ure 4.6). Despite the low signal levels, the KD of the interaction could reliablybe determined to be approximately 100 μM. Not only histamine interactedwith the β3 GABAAR, additionally 16 histaminergic ligands bound to the re-ceptor with KDs lower than 300 μM (Table 4.3). Surprisingly, although the KDof histamine was similar to that determined for histamine type 1 and 2 recep-
45
0 30
0
2
Time [s]
Respon
se[RU]
0 5 · 10−4 1 · 10−3
Concentration [M]
Figure 4.6. Sensorgrams and steady-state signals as a function of histamine concentra-tions for affinity determination of the interaction between histamine and β3 GABAAR.
tors [78], no other histamine type 1 receptor ligands were identified as ligandsof β3 receptors. From the ligands of the histamine type 2 receptor, only famo-tidine and tiotidine interacted with the receptor, binding of cimetidine was notdetected. As described above, the lack of cimetide binding might exclude β3GABAARs as candidates for the HisCl identified in neurons [47, 139]. Mostligands that were identified to bind to β3 receptors, are known to bind to his-tamine type 3 and 4 receptors. Specific ligands to the histamine type 4 recep-tor interacted with β3 GABAAR, making the histaminergic pharmacology ofthis receptor complete. Although the defined pharmacology seems unique forβ3 receptors, high-affinity ligands could not be discovered. Most histaminer-gic ligands bind to their designated histamine receptor with affinities that areabout two to three orders of magnitude higher than at β3 receptors (Table 4.3)[77, 78, 79, 31].
Competition of histaminergic ligands at unknown sites
One of the advantages of SPR biosensor technology represents a limitation atthe same time. For interaction analysis, structural information about the targetprotein is not required. When a ligand binds to the target protein, it is not ob-vious from the data where the ligand binds. If suitable reference compoundsand structural information of their binding sites are available, then competitionanalysis can indicate where the ligand binds, as described in Paper V. With-out such information, competition analysis can only indicate whether ligandscompete but not identify the binding site or resolve the type of competition.
Competition analysis was performed to study the possible competition be-tween histamine and the 16 histaminergic ligands interacting with the β3 re-ceptor. The assay that was used for competition analysis is illustrated in Fig-ure 4.7 and the results show that 13 of 16 histaminergic ligands compete withhistamine for binding to the β3 receptor. The identification of ligands thatcompete with histamine leads to a question that could not be answered yet,
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Table 4.3. Affinities of histamine and 16 histaminergic ligands to β3 GABAAR andcomparison with affinities at histamine receptors. hHR, human histamine receptor.
Ligand pKD Histamine receptor activity pKI at hHR
Thioperamide 4.9 H3/H4 antagonist 6.9/7.3JNJ7777120 4.6 H4 antagonist 7.84-Methylhistamine 4.5 H4 agonist 7.3Tiotidine 4.5 H2 antagonist 7.8Burimamide 4.5 H2/H3 antagonist, H4 agonist 5.4/7.9/7.4A-987306 4.3 H4 antagonist 8.3Imetit 4.3 H3/H4 agonist 8.8/8.2(S)-α-Methylhistamine 4.3 H3/H4 agonist 7.2/5.4VUF 8430 4.3 H4 agonist 7.5Clobenpropit 4.2 H3 antagonist, H4 agonist 8.6/8.1Immepip 4.2 H3/H4 agonist 9.3/7.7Famotidine 4.1 H2 antagonist 7.8Histamine 4.0 endogenous agonist 4.2/4.3/8.0/7.8Proxyfan 4.0 H3/H4 agonist 7.9/ 7.3A-943931 3.9 H4 antagonist 8.3(R)-α-Methylhistamine 3.7 H3/H4 agonist 8.2/6.6Iodophenpropit 3.5 H3/H4 antagonist 8.2/7.9
namely the type of competition. The ligands could compete directly at thehistamine binding site or at allosteric sites. Preliminary results from electro-physiological studies, performed at the Medical University of Vienna, weredifficult to interpret but indicate that histaminergic ligands bind to β3 recep-tors at different sites (unpublished).
In this study, a novel SPR biosensor assay was developed for investigat-ing the direct interactions of histaminergic ligands with homo-oligomeric β3GABAARs. The sensitivity and stability of the assay allowed to measuresmall molecule interactions, like histamine with a molecular weight of only111 g mol−1. Although the affinities of the histaminergic ligands interactingwith β3 GABAARs are too low to develop a high-affinity radioligand, the SPRassay can be used in the future to screen for such ligands.
4.5 Identification and characterization of novel AMPAreceptor protein interaction (Paper IV)
AMPA receptors are modulated by auxiliary proteins, such as transmembraneAMPA receptor regulatory proteins [126], cornichons [112] and shisa-9 [132,111]. These proteins modulate different aspects of AMPA receptor function,such as receptor gating, synaptic localization and surface expression, thereby
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Figure 4.7. Top: Illustration of assay for competition analysis. Bottom: Ligands thatcompeted with histamine at β3 receptors showed lower signals (green bars) when in-jected with histamine than the theoretical signal when no competition occurs (marks).
adding a further level of regulation that goes beyond the subunit assembly ofthe receptors [112, 126].
The novel interaction between Noelin1 and the AMPA receptor was stud-ied in Paper IV. Noelin1 is a secreted glycoprotein and has been shown to beimplicated in the development of the central nervous system in Xenopus lae-vis, chicken embryos and mice [6, 85, 1]. Proteomic analysis has identifiedNoelin1 as an AMPA receptor interacting protein [111]. The functional impli-cations of the Noelin1 interaction with AMPA receptors are still unknown. Ina first step towards such functional analysis, the interaction between Noelin1and AMPA receptors was studied using immunoprecipitation in mice brain,HEK cells and SPR biosensor based analysis.
4.5.1 SPR assay for protein interaction studies of AMPA receptorThe SPR biosensor assay for interaction studies between Noelin1 and theGluA2 AMPA receptor was designed essentially as described for the β3 re-ceptor (Figure 4.5). The assay development benefited from the previous studyaccelerating the identification of suitable solubilization and assay conditions.For this receptor, assay development was focused on affinity capture and anal-ysis in a solubilized state. Immobilizing the receptor by on-surface recon-stitution, as described in Section 3.4.1 [59], was not tested. The non-ionic
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detergent DDM was used for both solubilization and interaction analysis, andproved to by suitable for the protein-protein interaction studied. The samedetergent has been used for purifying GluA2 AMPA receptors for X-ray crys-tallization [119]. The affinity capture of the GluA2 receptor was performed es-sentially as for the β3 GABAAR, except that an epitope- and not a tag-specificmonoclonal antibody was used. However, similar results were obtained, andthe GluA2 AMPA receptor was specifically immobilized on SPR biosensorsurfaces, inferred from comparison of the immobilization levels from the re-ceptor and control surfaces.
4.5.2 Direct interaction of Noelin1-3 with GluA2 receptorOne of four Noelin1 isoforms was chosen for the present interaction analysis,namely isoform 3, referred to as Noelin1-3. This isoform was enriched in im-munoprecipitation assays performed in mice brain extracts and in postsynapticdensity fractions.
The direct interaction of Noelin1-3 and GluA2 AMPA receptors was shownby immunoprecipitation of HEK cell samples. Using SPR based interactionanalysis, the direct interaction was confirmed (Figure 4.8). The apparentkinetic rate constants were determined by using a 1-step interaction model(Scheme 3.1) to k1 = 2×106 M−1 s−1 and k−1 = 2×10−3 s−1, correspondingto an affinity of KD = 1nM. The data obtained in the SPR based experiments,together with data obtained from immunoprecipitation assays, strongly sug-gest that Noelin1-3 interacts directly with the GluA2 receptor.
The kinetic analysis of the interaction between Noelin1-3 and the GluA2 re-ceptor was challenging for several reasons. Noelin1-3 was only semi-purifiedprior to interaction analysis. Moreover, the protein concentration was deter-mined by masspectrometric analysis, where material can be lost during thepreparation. Both these practical difficulties can lead to an inaccurate estima-tion of the kinetic rate constants, since the analysis assumes that the proteinconcentration is well defined. Furthermore, the 1-step model employed fordata analysis did not capture the potential complexity of the interaction be-tween Noelin1-3 and the GluA2 AMPA receptor. The kinetic rate constantsare therefore approximations. Despite these limitations of the data analysis,the approach demonstrates the possibility to obtain kinetic information fromsemi-purified protein samples.
Another aspect that was investigated in this study was the interaction ofNoelin1-3 under reducing and non-reducing conditions. It has been shownthat the Noelin1-3 ortholog in mice, Pancortin 3, forms oligomers by disulfidebonds [1]. The interaction of Noelin1-3 was therefore studied in the pres-
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Figure 4.8. Top: Sensorgrams of interaction between Noelin1-3 and GluA2 AMPAreceptor. A 1-step model (Scheme 3.1) was fitted to experimental data (gray). Bottomleft: Sensorgrams of Noelin1-3 injected in the presence and absence of dithiothreitol,DTT. Bottom right: Signal levels extracted at the end of the injections (marks) ofNoelin1-3 with and without DTT (cycles within the same run are indicated).
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ence and absence of dithiothreitol. Under reducing conditions, no interac-tion between Noelin1-3 and the GluA2 AMPA receptor was detected, while itclearly occurred under non-reducing conditions (Figure 4.8). These results in-dicate that oligomerization of Noelin1-3 is required for interacting with GluA2AMPA receptors.
In addition to the biochemical and biophysical methods employed, im-munocytochemistry indicates that Noelin1 interacts with GluA2 AMPA re-ceptors at the synaptic level. Noelin1 co-localized in primary hippocampalneurons with extracellular matrix and postsynaptic density protein markers.
The study of the interaction between Noelin1-3 and the GluA2 AMPA re-ceptor using SPR biosensor technology provided kinetic information that can-not be obtained with in vivo and in vitro immunoprecipitation assays. The de-veloped assay can be employed for further studies of AMPA receptor auxiliaryproteins and potentially even for screening for small molecules that modulateits interactions with other proteins.
4.6 Fragment screening for identification of novelligands (Paper V)
The last study of this thesis involved the human immunodeficiency virus type1, the causative agent of acquired immunodeficiency syndrome. It serves asan outlook how fragment screening against ligand-gated ion channels could beperformed in the future.
HIV-1 RT, a key protein in the infection cycle of human immunodeficiencyvirus type 1, transcribes viral ribonucleic acid into deoxyribonucleic acid.Subsequent incorporation of the synthesized deoxyribonucleic acid into thehost genome enables proliferation of the virus. Inhibition of HIV-1 RT by nu-cleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reversetranscriptase inhibitors (NNRTIs) in combination with human immunodefi-ciency virus type 1 protease inhibitors has greatly improved the survival ofpatients infected with the virus [15]. NRTIs bind to the active site of the en-zyme and prevent the transcription of ribonucleic acid. NNRTIs bind to anallosteric pocket of HIV-1 RT and reduce the structural flexibility of the en-zyme , thereby preventing efficient reverse transcription [98, 15].
One of the major challenges of the treatment of patients with HIV-1 RTinhibitors, is the emergence of resistance mutations. Three common singlepoint mutations in the NNRTI binding pocket involve the replacement of ly-sine by asparagine at position 103 (K103N), tyrosine by cysteine at position181 (Y181C) and leucine by isoleucine at position 100 (L100I) [101]. The
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aim of the present fragment library screening was to identify novel scaffoldsthat bind to the NNRTI binding pocket of HIV-1 RT and inhibit the enzymevia this allosteric site. To account for drug-resistance, stringent criteria wereset that fragments should interact and inhibit wild type HIV-1 RT and the threementioned resistant variants (K103N, Y181C, L100I). The screening strategyis shown in Figure 4.9.
Figure 4.9. Strategy for fragment screening against HIV-1 RT. Regular boxes: SPRbased experiments, dashed boxes: enzyme activity assay. Rounded boxes show theoutcome of each screening step.
4.6.1 Primary screenDifferent strategies can be pursued for SPR based primary screens. In singleconcentration screens, the fragments are injected at a single concentration toidentify fragments whose signal levels are within a predetermined threshold[102]. Single concentration screens have also been performed in parallel withcompetition screens, using a known ligand to the target protein as a referenceligand, similarly as illustrated in Figure 4.7 [90]. In Paper V, the primaryscreen was performed by injecting fragments in 2-fold dilution series of fourconcentrations. This strategy is more laborious than a single concentrationscreen, but the advantage is that apparent affinities of the fragments can bedetermined. After the primary screen, 165 were selected and the number wasfurther narrowed down to 96 fragments after visual inspection.
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4.6.2 Competition and inhibition screenFrom the 96 fragments that were selected in the primary screen, 20 competedwith nevirapine. In parallel, the inhibition of the selected 96 fragments wasstudied in an enzyme activity assay. This revealed that 27 fragments inhibitedHIV-1 RT with IC50s lower than 1 mM. When comparing the results from theSPR based competition screen with those from activity based assay, 10 frag-ments were identified to compete and inhibit wild type HIV-1 RT. 10 frag-ments, both competing with nevirapine and inhibiting wild type HIV-1 RT,were subsequently subjected to affinity and inhibition quantification.
4.6.3 Quantification of affinity and inhibitionIn the last step of the fragment screening against HIV-1 RT, detailed affinityand inhibition quantification of three drug-resistant mutants (K103N, Y181C,L100I) and wild type HIV-1 RT were performed. Two fragments were ex-cluded as false positives. Of the remaining 8 fragments, fragment 4 showedthe most promising results. This fragment bound to all four HIV-1 RT variantswith μM KDs. It also inhibited the variants with μM IC50s. Sensorgrams offragment 4 with wild type HIV-1 RT and its structure are shown in Figure 4.10.
Figure 4.10. Sensorgrams of compound 4 (right) interacting with wild type HIV-1 RT.
4.6.4 Implications for fragment based drug discovery againstligand-gated ion channels
The fragment screen against HIV-1 RT shows aspects that should be taken intoaccount for future SPR based screening against ligand-gated ion channels.
One of the main challenges of discovery of drugs targeting ligand-gated ionchannels is their validation as drug targets (Section 2.2.3) [57]. HIV-1 RT is awell validated drug target and the positive therapeutic effects of inhibiting theenzyme are evident [15]. For ligand-gated ion channels, the situation is more
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complex. It is likely that drugs should act as modulators and not only as in-hibitors, like benzodiazepines and general anesthetics. For fragment screeningagainst ligand-gated ion channels, the use of SPR biosensor technology willrequire complementary methods to determine the type of modulation, like ra-dioligand binding assays and electrophysiological techniques. In comparisonto the relatively easy enzyme activity assay employed for inhibition studies ofHIV-1 RT, these methods are more elaborate, for example involving the use ofradioactively labeled compounds.
Although SPR biosensor technology has sufficient throughput for fragmentscreening, a limiting factor when screening against ligand-gated ion channelsmight be their stability on the sensor surface. However, the β3 GABAAR wasstable for more than 15 hours before a new surface was prepared (Paper III),thereby providing sufficient throughput for screening a fragments library inone replicate in one or two experiments.
The present study has benefited greatly from the availability of well char-acterized reference compounds, like nevirapine and delavirdine. Furthermore,there is detailed information about HIV-1 RT in apo structures and bound toinhibitors. Drug resistant variants are well defined and can be expressed andpurified similarly as with wild type HIV-1 RT. In contrast, crystal structures ofligand-gated ion channels are limited to a few examples, as described in Sec-tions 2.2.1 and 2.2.2. Such sparse structural information about ligand-gatedion channels, limits structure based drug discovery.
A technical aspect that needs to be taken into account, relates to the useof dimethyl sulfoxide for dissolving fragments, and its use in SPR based ex-periments. The concentration of dimethyl sulfoxide in the fragment screenagainst HIV-1 RT was 5 %, which was acceptable for this target. For the SPRbased interaction analysis of β3 GABAARs, a concentration of only 0.2 % wasemployed. At higher dimethyl sulfoxide concentrations, interactions with thereceptor could no be measured, presumably due to loss of structural integrity(unpublished).
Concluding this study, screening for novel scaffolds against HIV-1 RT byemploying SPR biosensor technology has led to the identification of a com-pound with micromolar affinity and potency at wild type and drug-resistantenzyme variants. Following up this study with SAR analysis of compound 4could eventually lead to a more potent lead. With respect to screening againstligand-gated ion channels, it seems that SPR based technology is not far awayfrom becoming a tool in drug discovery against these important drug targets.
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5. Future perspectives
There is a quote by Santiago Ramon y Cajal at the beginning of this thesis, stat-ing that the brain contains large stretches of unknown territory. The main goalin this thesis has been to uncover parts of the territory that relate to synapticprotein interactions. Different aspects of such interactions and that have impli-cations for both basic neuroscience and drug discovery were revealed. Study-ing the relationships between synaptic proteins is required for an improvedunderstanding of basic brain functions, like Ca2+ signaling and modulation ofligand-gated ion channels involved in synaptic plasticity. In addition, provid-ing tools to discover novel therapeutics that target ligand-gated ion channelsis of importance for accounting for the increasing burden of neurological dis-orders for patients and health care.
Novel synaptic protein-protein interactions were identified and character-ized by SPR biosensor based analysis. The combination of biophysical, bio-chemical and biological methods, has proven to be suitable for such anal-ysis. For similar studies in the future, it is important to recognize that theinvestigated proteins do not necessarily need to be pure to perform biosensorbased interaction analysis. Instead crude lysates from transfected cells can bescreened with respect to their binding to the target protein in a relatively shortamount of time. After such preliminary analysis, the most promising proteinscan be investigated in a more defined environment. Such a strategy is espe-cially useful for confirming and characterizing the interactions of auxiliaryproteins with the AMPA receptor. These proteins can be membrane bound orrequire oligomerization, which makes it more difficult to retain their stabilityduring purification.
Over the last years, FBDD by SPR has emerged as a successful strategy toscreen for novel ligands against GPCRs. For drug discovery against ligand-gated ion channels, another class of important drug targets, a sensitive andstable SPR biosensor assay has been developed. It can be expected that simi-lar assays will be employed in the future, either for ligand-gated ion channelsor other classes of ion channels. Thereby, the primary challenge is to pro-duce oligomeric ion channels with a defined subunit composition in sufficientamounts. With the availability of such proteins, the development of an SPRbiosensor assay, although technically challenging, should not be the limitingfactor towards fragment screening against ion channels.
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6. Sammanfattning
I var hjarna finns det miljarder av nervceller, sa kallade neuroner. Dessa kom-municerar med varandra genom synapser, stationer dar information fran enneuron kan overforas till en annan. I varje synaps finns hundratals proteinersom interagerar med varandra. Dessa interaktioner kontrollerar synapsernasfunktion och, pa en hogre niva, aven hjarnans. For att fa en battre forstaelseav hur hjarnan fungerar, ar det darfor viktigt att forsta dessa interaktioner.
I den har avhandlingen har interaktionerna mellan olika synaptiska pro-teiner studerats med hjalp av ytplasmonresonans biosensorteknologin. Det aren kanslig teknik som gor det mojligt att studera interaktioner over tid i endefinierad in vitro omgivning. Det ger detaljerad information om interaktio-nens kinetik. Denna detaljerade karaktarisering gor att man kan fa en battreforstaelse for de synaptiska proteinernas funktion i hjarnan.
I artikel I och II har proteiner som ar involverade i synapsernas Ca2+ signa-lering studerats. Calmodulin och calendrin ar tva Ca2+-bindande proteiner ochAKAP79 ar ett sa kallat scaffolding protein, som interagerar och forankrar an-dra proteiner pa ratt stalle i synapsen, sa att de kan utfora sitt regulatoriska ar-bete. Man visste sedan tidigare att AKAP79 interagerar med calmodulin menatt den aven interagerar med caldendrin har identifierats i artikel I. Caldendrinoch calmodulin interagerar med AKAP79 pa samma stalle i proteinet, menpa helt olika satt. Calmodulin binder bara till AKAP79 i narvaro av Ca2+,vilket ar typiskt for detta protein, medan caldendrin binder till AKAP79 badei narvaro och franvaro av Ca2+. Jonernas funktion vid signalering ar att reglerafunktionen hos olika proteiner. Finns det tillgang till Ca2+ binder calmodulintill andra proteiner, och tvartom, finns det inget eller bara en lag koncentra-tion, forhindras calmodulins interaktioner till andra proteiner. Caldendrin aandra sidan, binder AKAP79 bade med och utan Ca2+, aven om interaktionenar svagare i franvaro av Ca2+. Darfor ar det troligt att calendrin har en annanfunktion an calmodulin i synapserna.
En viktig klass av proteiner i synapserna ar ligandstyrda jonkanalerna. Desitter i det postsynaptiska membranet och oppningen av jonkanalen styrs avinteraktionen med en signalsubstans. Da jonkanalen ar oppen strommar jonergenom kanalen vilket gor att membranpotentialen andras. De inhibitoriskajonkanalerna gor att membranpotentialen blir mer negativ vilket i sin tur in-hiberar signaloverforingen. De exitatoriska jonkanalerna har motsatt effekt. I
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artikel III och IV har tva ligandstyrda jonkanaler studerats, den inhibitoriskaGABAA receptorn och den excitatoriska AMPA receptorn.
I artikel III utvecklades en biosensor assay for att kunna studera interak-tioner av sma molekyler med β3 GABAA receptorn, som bestar av fem iden-tiska β3 subenheter. Receptorn har inte identifierats i hjarnan an, men manformodar att den finns. For att identifiera β3 receptorn i hjarnan, behovs deten unik farmakologi, allsta substanser som binder till receptorn pa ett uniktsatt. Genom att screena ett substansbibliotek, har det identifierats en unik far-makologi. For att kunna hitta β3 receptorn i hjarnan, sa behovs ytterliggaresubstanser som har hogre affinitet an de, som har identifierats. Den biosensorassay som i denna studie har utvecklats, kan i framtiden anvandas till att ly-ckas identifiera substanser med hog affinitet till β3 receptorn.
AMPA receptorn ar en excitatorisk jonkanal och involverad i synaptisk plas-ticitet av hjarnan, som ar grunden for att bygga minnen. Funktionen av AMPAreceptorn ar regulerat via andra synaptiska proteiner. I artikel IV har en nyinteraktion mellan Noelin1 och AMPA receptorn identifierats och karaktaris-erats med hjalp av en biosensor assay. Funktionen av denna interaktion arhittills okand, men det pagar funktionella studier som ska ge ett svar pa detta.
Den sista studien (artikel V) handlar inte om ett synaptisk protein, utanistallet om ett nyckelprotein hos HIV, omvant transkriptas. Denna studie gerett exempel pa hur fragment baserad lakemedelsutveckling kan anvandas foratt identifiera nya lakemedel mot ligandstyrda jonkanaler. Ett bibliotek av1014 fragment, som ar sma organiska molekyl, screenades mot vildtypen ochtre resistenta varianter av omvant transkriptas, och tre resistenta varianter.Efter flera steg, dar bade biosensortekniken och en aktivitetsassay har anvants,blev en substans identifierad som band till och inhiberade alla 4 proteinvari-anterna. Denna studie utgor darmed en intressant utgangspunkt for utvecklingav nya lakemedel mot HIV.
Den har avhandlingen visar hur detaljerad interaktionsanalys kan bidra tillen battre forstaelse for interaktionerna mellan synaptiska proteiner och derasfunktioner. Ytplasmonresonans biosensorteknologin har bidragit med viktigkinetisk information och visar sig vara lampad for framtida fragment baseradlakemedelsutveckling mot ligandstyrda jonkanaler.
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7. Zusammenfassung
Das menschliche Gehirn ist ein fantastisches und hoch komplexes Organ. DasVerhalten wird zum großten Teil durch die Zusammenarbeit von ungefahr 100Milliarden Nervenzellen bestimmt, den Neuronen. Um Signale von einemNeuron zu einem anderen ubertragen zu konnen, benotigt es spezielle Schalt-stellen. Diese sind die Synapsen, die aus der Prasynapse, dem Sender, einesNeurons und der Postsynapse, dem Empfanger, eines anderen Neurons beste-hen. In jeder Synapse gibt es Hunderte von Proteinen, die miteinander wech-selwirken und damit die Funktion unseres Gehirns entscheidend beeinflussen.Aus diesem Grund ist es wichtig die Wechselwirkungen der synaptischen Pro-teine zu untersuchen, um damit ein besseres Verstandnis der Funktion desGehirns zu erreichen. Diese Doktorarbeit handelt von Studien der dynamis-chen Vorgangen (Kinetik), die die Wechselwirkungen von synaptischen Pro-teinen entscheidend beeinflussen. Fur diese Studien wurde Oberflachenplas-maresonanz Spektroskopie angewendet, um die Wechselwirkungen in Echtzeitund in einer kunstlichen Umgebung studieren zu konnen.
Ca2+ ist ein wichtiger Botenstoff in unserem Korper und vor allem imGehirn, wo es entscheidend zur Signalubertragung beitragt. Diese Effektewerden durch Ca2+-bindende Proteine, zum Beispiel Calmodulin und Calden-drin vermittelt, die in den Artikeln I und II undersucht wurden. Genauergesagt, wurden deren Wechselwirkungen mit dem Gerustprotein AKAP79studiert. AKAP79 verankert andere Protein an der richtige Stelle innerhalbder Synapse, so dass diese ihre Funktionen richtig ausfuhren konnen. Calden-drin und Calmodulin binden an einer ahnlichen Stellen zu der Proteinstrukturvon AKAP79, dennoch sind deren Wechselwirkungen sehr unterschiedlich.Calmodulin kann AKAP79 nur in der Anwesenheit von Ca2+ binden, wastypisch fur dieses Protein ist. Diese Regulierung ist ein relativ einfacher Weg,um die Funktion eines Proteins durch die Konzentration eines kleinen Ions,wie Ca2+, an- und auszuschalten. Auf der anderen Seite, so konnte Calden-drin mit AKAP79 sowohl in der Anwesenheit als auch der Abwesenheit vonCa2+ wechselwirken. Diese Eigenschaft zeigt, dass Caldendrin in der Synapseandere Funktionen ausfuhrt, die nicht so sehr von der Ca2+ Konzentrationabhangig sind, wie die von Calmodulin.
Ionenkanale spielen fur die Signalubertragung in dem Zentralen Nerven-system eine wichtige Rolle. Durch die Bindung eines Signalmolekuls wirdder Ionenkanal geoffnet und Ionen konnen durch die Zellmembran fließen.
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Der Ionenfluss hat einen großen Einfluss auf die Signalweiterleitung in denSynapsen. Inhibitorische Ionenkanale, wie der GABAA Rezeptor, hemmendie Weiterleitung von Signalen, wahrend exzitatorische Ionenkanale, wie derAMPA Rezeptor, die Weiterleitung ermoglichen. Die Balance zwischen Hem-mung und Aktivierung ist eine Voraussetzung fur das die korrekte Funktionunseres Gehirns. Abweichungen von dieser Balance konnen schwerwiegendeFolgen nach sich ziehen und zu neurologischen Erkrankungen fuhren.
Der Ionenkanal, der in Artikel III studiert wurde, ist ein GABAA Rezep-tor, der aus 5 identischen β3 Proteineinheiten aufgebaut ist, und noch nichtim Gehirn nachgewiesen werden konnte. Fur den Nachweis benotigt es Sub-stanzen, die in einer spezifischen Weise an den Rezeptor binden. In dieserStudie wurden solche Substanzen entdeckt. Was aber noch benotigt wird, umden β3 im Gehirn nachzuweisen, ist eine oder mehrere Substanzen, die nochstarker an das Protein binden, als die die entdeckt wurden. Die Methode, diewahrend dieser Studie entwickelt wurde, kann aber in der Zukunft von großerHilfe sein, um solche Substanzen zu identifizieren.
Ein exzitatorischer Ionenkanal wurde in Artikel IV untersucht. Dieser soge-nannte AMPA Rezeptor ist wichtig fur die Plastizitat unseres Gehirns, was dieBasis ist um Erinnerungen aufzubauen. Die Funktion dieses Ionenkanals wirdin den Synapsen von Helferproteinen beeinflusst und in dieser Studie wurdedie Wechselwirkung eines Proteins, Noelin1, mit dem AMPA Rezeptor zumersten Mal nachgewiesen. Uber die Funktion von Noelin1 kann momentannur spekuliert werden, aber es scheint, dass dieses Protein haufig wahrend derEntwicklung des Zentralen Nervensystems auftritt.
Die letzte Studie (Artikel V) handelt nich von einem synaptischen Pro-tein, sonder von der reversen Transkriptase, ein Schlusselprotein von HIV.Diese Studie dient als Beispiel wie mit Hilfe von Fragment-basierter Phar-maforschung Medikamente entdeckt werden konnen, die die Funktion vonIonenkanalen beeinflussen und zur Behandlung neurologischer Krankheiteneingesetzt werden konnen. Eine Sammlung von Fragmenten, kleine organis-che Molekule, wurden gegen den Wildtyp der reversen Transkriptase und 3resistenten Varianten getestet. Dabei wurde eine Substanz gefunden, die dieFunktion aller 4 Proteine hemmt, und dadurch einen interessanten Startpunktfur zukunftige Pharmaforschung gegen HIV darstellt.
Diese Arbeit zeigt, wie detaillierte Analyse der Wechselwirkungen vonsynaptischen Proteinen zu einem besseren Verstandnis dieser Proteine un derenFunktion fuhren kann. Die angewandte Oberflachenplasmaresonanz Spek-troskopie hat dabei wichtige kinetische Informationen geliefert und ist dafurgeeignet fur die Pharmaforschung und die Suche nach neuen Medikamenteneingesetzt werden, die die Funktion von Ionenkanalen beeinflussen.
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8. Acknowledgments
A few years ago, my primary goal was to become a faster cross-country skier.But I realized that being a PhD student is quite similar to skiing competitions,and I never regretted my decision to replace skis by pipettes. An importantrequirement for success in either area is to be part of a team that creates an en-vironment that allows one to push the own limits. I am very thankful to haveencountered such an environment at the Department of Chemistry.
Helena Danielson, thank you for the opportunity to become a PhD studentin your research group and at Beactica. It was a pleasure having you as a su-pervisor. Your support, not only related to my research but also in personalmatters was invaluable. I enjoyed your guidance but also the freedom to de-velop my independence as a scientist.
Mikael Widersten, thank you being my co-supervisor and your support.More importantly, for encouraging me to keep using Linux. Tanks to you,I am even giving OpenSUSE a second chance. Keep encouraging others andstay open source.
I want to thank my co-authors for their contribution to this thesis. WernerSieghart, your knowledge about GABAA receptors is impressive and I amthankful for your patience and kindness when sharing it with me. NikhilPandya, it is great to work with you and to discuss science on Skype. Youare not only smart and helpful, you are a good colleague and always up forgoing out at our meetings. Xenia Gorny, thank you for our collaboration andfor visiting us in Uppsala, you have done a great job.
Current and previous team members of the HD group, with you, it was apleasure to be in the lab. Angelica, I can hear you coming to the office fromfar away, either by the tick tack of your shoes or your distinct laugh. To followyour development up to the point where you defended your thesis was inspir-ing. It is still a mystery for me how you could manage all that work and stillhave time for all your sessions in the Dr. Ehrenberg therapy chair. And notonly that, you are on the best way to an alternative career as a langloppsakare,I am expecting you to break the 2:00 hour mark at next years Tjejvasan. Eldar,I am very glad to have met you and I am impressed by your calmness whensupervising many different students. Whenever someone new is joining ourlab, then I mostly hear “Eldar will take care of the student.” Good luck for
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the rest of your PhD and enjoy the Formula 1 season. Sara Solbak, you areadding Norwegian to the multiple languages in the lab. I do not understandeverything you say but it sounds really nice. Vladimir, I hope to get to knowyou better in my last weeks in the lab. Good luck for your PhD. Tony, thankyou for supervising me during my first steps in the lab as a PhD student and forour climbing trips to the locals crags. Your calmness was a welcome contrastto the pushy atmosphere that we sometimes encountered. Sofia, when you arearound, there is a aura of happiness and Swedish royalism. From you, I gota Dr. title long before I even defended my thesis, which I am very proud of.Thank your for initiating the pHD film project - may hats and songs not returnto our disputation parties. Johan Winquist, thank you for being the helpful,supportive and glad person that you are. I enjoyed our training sessions inthe gym and on the bike. Helena Nordstrom, with you I could discuss the tiny,tiny details of our work. For the future I wish you all the best and. . . stay warm.
I consider myself very lucky to have been an industrial PhD student at Be-actica. Per, you are such a kind and supporting colleague, thank you for thegreat opportunity to be part of the Beactica team. I appreciate especially thatyou made me think out of the academic box on several occasions. Malin, theenergy that you spread is almost tangible and your sprints through the B7:3corridors are legendary. It was great fun to work with you and to discuss howto correctly use a measuring cylinder. And of course, thank you very much forthe precious ME proof reading. Gun, I enjoyed sharing the office with you andour discussions about the goods and bads of the scientific world. Thank youfor letting me take part of your wealth of experience. Maria, I was happy tomeet you again at Beactica, after we met years before at the EBC. Ways crosswhen you least expect it and I look forward to the next time our ways willcross. Mari, I enjoyed working with you in the last phase of my PhD studies.Your persistence when it comes to ensuring quality in the lab is admirable, andI promise, I will label my boxes properly. Matthis, stubborn to bike in wind,rain and snow, thank you for introducing me to the world of SPR biosensors,it is a fantastic one. Thomas, you perfectly exemplify the idea of first-think-then-speak. Your calmness is inspiring and I wish you the best for your nextVasaloppet. Evert, besides your support to get started with molecular model-ing, I will mainly remember you for introducing me to long-distance skating,thank you, was it such an experience. Peter, thank you for expert help withExcel and your tasty Sushi, domo arigato.
Thank you to more biochemists from the B7:3 corridor. Gunnar Johansson,as a professor you combine kindness with deep curiosity for science, a wel-come combination. Thank you for your spontaneous greetings in our movies,they were highly appreciated by the audience. Erika, as a director, you haveproven to be a worthy successor and you have shown great skills on rollerskis. I hope that Angelica’s movie was not your last. Good luck for your PhD
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and as a spinning instructor. Marcus, thanks for online cheering during myfirst Vasaloppet, I will let you know when I will be at the start again. Doreen,when I was most stressed, your encouraging words were more than welcome.If you say so, then I will manage my defense. Dirk, it is great to know youand to have a Badisch speaking colleague. Your German precision becomesevident daily when, at 11:45, it is time for “lunch?”. I hope we can go climb-ing again, I promise, you will not always have sore muscles for a week. Ylva,thank you for appreciating me being “German” sometimes, I hope you havefun with your brand new gel casts. Gustav, soon you will have been a PhDstudent for 18 weeks, a great occasion to celebrate. Just for clarification, Ihave a LATEX fetish but not a Latex fetish. Anna Tornsten, as a regular guest atdisputation parties, I hope that I can welcome you to mine as well. Therese,thank you for your great effort towards a clean corridor and bioreactor lab.
To the the B7:4 biochemists. Cissi, you rowed twice at the AkademiskaRodden, you machine. Thank you for calming me down when I get toostressed. Asa, I just wanted to say “Hej”, as this is what we usually do when wemeet on the bike. Good luck on the finishing straight towards your defense.Emil, it is always a pleasure to have an elevator-chat with you. Huan, yourdancing in “Tony Style” is one of the most funny scenes in our movies, welldone. Paul Bauer, somehow you seem to be still part of the MW group. Youare a true computational chemist with Guru-like IT knowledge. Francoise, Iknow that you do not like to speak in front of a camera, but be assured, youare doing great. Your encouraging words about my writing have been morethan welcome. Mikael “Nisse” Nilsson, I am hoping for at least one commonclimbing session before I leave.
Enough of thanking biochemists, now it is time for. . . Sara Norrehed, youhave tried hard but I did not dare to change the dedication of the thesis. Thankyou for giving my figures a touch of Norrehed. Magnus, go for gold. Fol-lowing your comments during Olympic games or World Championships ismore exciting than the competitions (“Saaaaa jaaa!!!”, “Kalla for f. . . ! Guuu-uuld!!!!”). Christian, what a great name you have. You wanted to try AllgauerKasespatzle? Here you go: fry lots of onions in lots of butter in a pan. Makea dough with eggs, flavor, water, salt. Make Spatzle with the special press,put lots of Allgauer Bergkase between layers of Spatzle and pour the fried andjust slightly greasy onions over, bon appetit. Rikard, great to have shared theexperience with you of organizing and cleaning-up the Fairy-tale party. To-bias, great to meet you at the lunch table. Johan Verendel, it seems that yourfairy costume has not only made an impression on me, it really suited you.Johanna Johansson, thank you for keeping my office chair warm, when I wasnot around. I am not sure whether you still follow my training advice, so hereis the most important one for your: take it easy. Johanna Andersson, it wasgreat to climb with you, keep on bouldering. Inger, you know which corridor
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has the better coffee machine. Thank you for taking care of all the bureau-cratic formalities. Gunnar Svensson, with you, Christmas comes every daywhen you bring us our presents. Bo “Bosse” Fredriksson, your help with ourcomputers is invaluable.
It is quite common for German students to come to Uppsala for an intern-ship and then they usually never want to leave. For me it was similar, andmy time in Uppsala began at the EBC. Siv Andersson, it must have been fatethat I found your picture showing you with your cross-country skis, when Iwas searching for an internship in Uppsala. Thank you for allowing me tobe part of your group, it was a summer that I will never forget. Kasia, youremember my pink fingernails after I lost a bet. I remember us digging for soilin the Stadskogen and our great time in the lab. You are truly inspiring andI envy you for working with Mammoth and Otzi, proteins seem really boringcompared to that. Hans-Henrik, you were the third member of the Stadsko-gen digging project, not bad for a bioinformatician. Thijs, I look forward toour next lunch discussion about the latest developments in the doping world.Fredrik Granberg, enjoy the reading and your next trip to Japan.
I want to thank the Uppsala climbing and mountainbike people, Shawn Boyeand family, Martin, Maria, Ruxandra, Veera and Pablo, for the fantastic hourswe spent climbing our favorite crags and riding single trails. Stort tack tillmina favorit skidakar tjejer, Sara-Lina och Yasmin, det har varit kul att traffaer och kunna lara er lite om skidakning och vallning. Jag ska aldrig glommastrumpor-over-tights stilen och att se en Duracell hare pa skidor.
Es gibt Personen, die mir bei meiner Arbeit geholfen haben. Aber wichtigerseid Ihr, die bei mir sind, egal wie gut meine Ergebnisse sind. Mama undPapa, egal welche Entscheidung ich getroffen habe, ob es ums Langlaufenging, ums Studieren, meine Doktorarbeit oder um meine Beziehungen, ihrhabt mich immer unterstutzt aber nie gedrangt. Fur diese Freiheit und diesenRuckhalt bin ich euch sehr dankbar. Sarah, Judith und Meli, ihr seit drei superSchwestern. Danke fur euren Kaffeekocher, er war bis zum Schluss ein treuerWachmacher, denn egal wie stressig es war, fur eine Tasse selbstgemahlenenKaffee habe ich mir daheim immer Zeit genommen. Maxi, du bist ein sehrguter Freund und ich freue mich schon auf Karpathos, Surfen, blutige Handeund gemutliche Abende. Siggi, die Tatsache, dass ich diese Arbeit in LATEXgeschrieben habe, liegt zum großten Teil an dir. Danke fur deine Hilfe, wennes um Laptops und ahnliches geht, und dafur ein guter Freund zu sein. Sara,ich hoffe, dass ich bei der Feier an Silvester keine Kaffeeflecken in die Mobeleingebrannt habe. Ich freue mich schon, euch in Uppsala zu begrußen.
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