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Structural and biochemical characterization of Roco proteinsTerheyden, Susanne

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Chapter 7

Summary and Discussion

Nederlandse Samenvatting

Acknowledgements

Susanne Terheyden and Arjan Kortholt

149

Summary and Discussion

Leucine-rich-repeat kinase 2 (LRRK2) is an extremely large and complex multi-domain

protein which turned out to be incredibly difficult to study in many different aspects.

Mutations in LRRK2 account for the majority of familial Parkinson’s Disease (PD) cases

(Pringsheim et al., 2014). Many LRRK2 mediated pathways and interaction partners have

been identified, but the cellular functions of LRRK2 and its malfunction in PD are still not

understood in great detail (Boon et al., 2014; Wallings et al., 2015; Roosen and Cookson,

2016; Rosenbusch and Kortholt, 2016; Tang, 2016b). PD-linked mutations in LRRK2 are

found in almost every domain, but are primarily located in the catalytic core of the protein

(RocCOR-kinase domains) (Cookson and Bandmann, 2010). Several of the PD-mutations

have been linked to a decrease in GTPase and/or an increase in kinase activity (West et al.,

2005; Greggio et al., 2006; Guo et al., 2007; Jaleel et al., 2007; Lewis et al., 2007; Li et al.,

2007; Luzón-Toro et al., 2007; Anand et al., 2009; Liao et al., 2014; Rudi et al., 2015b; Ho

et al., 2016). However, the molecular mechanisms of G-protein and kinase activation

remain to be determined. Here, our approach was to dissect the problem into smaller

pieces, e.g. the kinase domain or the RocCOR tandem/LRR-RocCOR, and approach one

after the other. My thesis was aimed to investigate the RocCOR domain tandem as the

central and name giving part of Roco proteins.

Several lines of evidence suggest that the nucleotide binding state (GDP/GTP) of the Roc

domain is important for kinase activation (West et al., 2005; Ito et al., 2007; Biosa et al.,

2013). Nevertheless it is not clear how the Roc domain regulates the kinase domain on a

molecular level. Classical G-proteins are inactive in the GDP-bound state and active when

GTP is bound. The switch regions are flexible in the GDP-bound state but will be fixed in

an active conformation when bound to the γ-phosphate of GTP, and thereby effectors can

bind to this region (Vetter and Wittinghofer, 2001). For LRRK2, the situation was not

clear: Liao et al. suggested that also the GTP bound form of the Roc domain is the active

conformation that can stimulate LRRK2 kinase activity (Liao et al., 2014). However,

several other studies showed that LRRK2 kinase activity does not change upon addition of

GDP, GTP, or non-hydrolysable GTP analogues (Liu et al., 2011a; Taymans et al., 2011),

while others suggested that an intermediate state during hydrolysis presents the active state

of LRRK2 (Biosa et al., 2013; Rudi et al., 2015b). Also other aspects of the regulation of

Roco protein besides RocCOR is still under debate (Nixon-abell et al., 2016).

150

Dimerization seems to be a major regulator of the Roco proteins’ G-protein cycle

(Gotthardt et al., 2008; Gasper et al., 2009) and LRRK2 in particular (Berger et al., 2010;

Daniëls et al., 2011b; Rudi et al., 2015b). Since biochemical evidence on this topic is

limited and the LRRK2 protein is very difficult to work with in vitro, we employed

prokaryotic Roco proteins as a model in order to study the biochemical and structural

features of the RocCOR domain tandem.

In Chapter 2 and 3 we investigated the influence of dimerization on Roc activity. We

confirmed that the C-terminal subdomain of COR (COR-B) is the dimerization device and

that dimerization is important for the GTPase activity but not GDP or GTP binding

(chapter 2, (Gotthardt et al., 2008)), highlighting the importance of dimerization for the G-

protein cycle. In chapter 3, we could show that the Roco protein from Chlorobium tepidum

(Ct) cycles between a monomer and dimer within half a minute in a nucleotide dependent

manner, which is in the catalytically relevant time scale for GTP hydrolysis (10 minutes)

(Deyaert et al., 2017a), implying that dimerization might have an important role in the

hydrolysis mechanism. A mutant homologous to one mutated in LRRK2 PD patients

(L487A, L1371V in LRRK2) shows impairment in this monomerization/dimerization cycle

and a reduction in the single turnover GTP hydrolysis rate.

Both chapters show that that dimerization is important for the G-protein cycle. However

the kinetic properties of the Roco G-protein cycle were not studied in great detail.

Therefore, we set out to characterize several Roco proteins including LRRK2

biochemically in a systematic fashion (chapter 4). Consistent with previous theories and

data (Gotthardt et al., 2008; Liao et al., 2014) we could confirm that all Roco proteins have

nucleotide affinities in the micro-molar range, meaning that they don’t need a GEF for

nucleotide exchange in contrast to classical small G-proteins. Moreover, we showed that

the KM of all Roco proteins is consistently in the higher micro-molar range, enabling them

to act as GTP sensors. Whether this is an important sensing function or just a kinetic

feature of the protein remains to be shown in the context of the cell. Additionally the large

difference between KM and KD points towards a more complex hydrolysis mechanism. We

could show that this difference is a feature of the hydrolysis reaction itself and that Pi

release is not the rate limiting step of the GTPase reaction. There seems to be a GTP

dependent mechanism involved, independent of the canonical binding/hydrolysis site that

we do not understand yet. All in all this shows that Roco proteins follow a unique G-

protein cycle, different from classical G-proteins. Moreover, for LRRK2 it has been

151

demonstrated that the kinase is stimulated only in the presence of GTP but not GppNHp (a

non-hydrolysable GTP analogue) or GDP, again indicating that no classical active or

inactive conformations are present but rather that the cycling itself is the active form that

enhances kinase activity.

Consistent with the hypothesis that not the GDP or GTP state is the active form, the

structures of the Mb RocCOR tandem in the GppNHp and GDP bound states show no

major differences in the switch region in contrast to conformational changes reported for

classical small G-proteins such as Ras (Chapter 5). This again points out the difference to

the classical small GTPases. Moreover we learned from these structures that the three

subdomains (Roc, COR-A and COR-B) can obtain multiple conformations relative to each

other. Also it seems clear that switch II and the RocCOR interface has an important role in

the activation mechanism and function of the RocCOR domain tandem. Despite the fact

that Mb RocCOR does not monomerize upon GTP binding, HDX experiments revealed

that it undergoes a similar conformational change as the Ct Roco.

Taken all this data together we suggest the following activation mechanisms for Roco

protein, here shown in the context of LRRK2 (Figure 1): Considering the nucleotide

affinities, the majority of the LRRK2/Roco protein should be GTP bound. Exchange from

GTP to GDP (and vice versa) is fast, but since the GTP concentration is usually 10 times

higher than GDP, all protein should be bound to GTP (Traut, 1994). Moreover it has been

demonstrated that the protein exists as a monomer in the cytosol and as a membrane bound

dimer which is the more (kinase) active fraction (chapter 6). The cytosolic monomer is

probably maintained by other proteins, namely 14-3-3. Recruitment to membranes is

regulated by binding of Rabs to the N-terminus (Liu et al., 2017). At the membrane

LRRK2 is a dimer and has its highest kinase activity. To be able to perform a hydrolysis

reaction, the Roc domains need to come together by which the COR domains probably

need to undergo a conformational change. Switch II and the hydrophobic interface between

the Roc and the COR domain are very important for this process to mediate changes in the

dynamic properties of the protein. In Ct it is possible that in order to allow this process, the

COR domain needs to dissociate (chapter 3). The hydrolysis mechanism probably works in

several steps, since we cannot explain the difference in KM and KD with a simple one step

hydrolysis mechanism (chapter 4). Here, more research is clearly needed to answer during

which step dimerization plays a role. As demonstrated in chapter 4, LRRK2 needs to

hydrolyse GTP in order to fully activate its kinase. It is possible that membrane bound

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LRRK2 has a higher GTP hydrolysis rate than the one we measured in vitro in solution as

a dimer. Localization, dimerization and also interaction with other proteins and

phosphorylation are important regulatory processes that influence each other and both

kinase and GTPase activities.

With this thesis we could give a first combined insight into kinetic, structural and

biochemical properties of the G-protein cycle of LRRK2 and Roco proteins. However, my

work also raised important new questions. Since Roco proteins have only a moderate

GTPase activity, it will be important to identify co-regulators; can for example membrane

binding stimulate both GTPase and kinase activity? Also the dynamic changes of the

protein especially in the RocCOR tandem need to be understood in order to explain the

complex crosstalk of Roc and kinase domain via COR and the effect of mutations in this

region. With the advances in the production of high quality LRRK2 protein, I believe it is

now possible to tackle at least some of these questions employing LRRK2 full-length

protein. Notwithstanding the important progress with LRRK2, still the panel of now

available prokaryotic Roco proteins is still a valuable addition and, as demonstrated here,

can give precious general insights and help to advance the field.

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Figure 1: Proposed activation mechanism of LRRK2.

154

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Nederlandse Samenvatting

De ziekte van Parkinson is een progressieve motorische aandoening, veroorzaakt door het

verlies van dopaminerge neuronen in de middenhersenen. Algemeen bekende symptomen

van Parkinson zijn tremor, stijfheid en posturale instabiliteit. Op cellulair niveau wordt

Parkinson gekenmerkt door de formatie van eiwitaggregaten. Deze zogenaamde “Lewy

bodies” bestaan uit α-synucleïne, Leucine Rich Repeat Kinase 2 (LRRK2) en andere

eiwitten. Ongeveer 2% van de personen ouder dan 80 jaar wordt wereldwijd getroffen door

Parkinson. De meeste gevallen zijn sporadisch (zonder bekende oorzaak), maar genetische

studies hebben aangetoond dat ten minste 10% van de gevallen verklaard kan worden door

Mendeliaanse erfelijkheid. Parkinson-geassocieerde mutaties zijn gevonden in

verschillende genen, waaronder SNCA / a-synucleïne, PINK1, LRRK2 en DJ-1. Het meest

frequent gemuteerde gen is LRRK2, dat autosomaal dominante vormen van Parkinson

veroorzaakt. Interessant is dat de symptomen van LRRK2 en sporadische Parkinson zeer

vergelijkbaar zijn en daarom zou inzicht in de LRRK2-functie kunnen helpen bij het

begrijpen van de ziekte van Parkinson in het algemeen.

LRRK2 is een zeer groot en complex eiwit dat uit meerder domeinen is opgebouwd

en daarom erg lastig te onderzoeken is. Ondanks dat veel LRRK2 interactiepartners zijn

geïdentificeerd, begrijpen we de cellulaire functies van LRRK2 nog steeds niet volledig.

LRRK2 heeft twee enzymatische domeinen, een GTPase en een kinase domein. De meest

voorkomende Parkinson mutaties hebben een verlaagde GTPase en verhoogde kinase

activiteit. Het is echter onbekend hoe het G-domein en kinase precies geactiveerd worden

en hoe de Parkinson mutaties de activiteit beïnvloeden. In dit proefschrift heb ik me

gericht op het onderzoeken van het moleculaire activeringsmechanisme en functie van het

G-domein. Omdat LRRK2 eiwit slechts in kleine hoeveelheden te isoleren is, heb ik ook

gebruikt van de homologe Roco eiwitten van lagere organismen als een model om de

biochemische en structurele aspecten van het RocCOR domein tandem te bestuderen.

LRRK2 behoort tot de Roco eiwitfamilie dat gekenmerkt wordt door de

aanwezigheid van een RocCOR domein tandem. Roc is het G-domein dat de GTPase-

activiteit heeft. Om de juiste functie te vervullen zijn G-nucleotiden (GDP en GTP)

essentieel. Ook dimerisatie, het samen komen van twee identieke eiwitmoleculen, is

belangrijk voor de functie van LRRK2. Hoofdstuk 2 en 3 van dit proefschrift benadrukken

het belang van dimerisatie voor de G-eiwit cyclus. Doormiddel van structurele en

biochemische studies konden we bevestigen dat het C-terminale deel van COR essentieel is

158

voor dimerisatie. Dimerisatie is belangrijk voor de GTPase-activiteit, maar niet voor GDP

of GTP-binding.

In hoofdstuk 4 hebben we verschillende Roco eiwitten, waaronder LRRK2, op een

systematische manier biochemisch gekarakteriseerd. In overeenstemming met eerdere

theorieën en gegevens konden we bevestigen dat alle Roco eiwitten een unieke G-eiwit

cyclus doorlopen. Ook laten we voor LRRK2 zien dat de kinase activiteit alleen

gestimuleerd is in de aanwezigheid van GTP maar niet GppNHp (een niet-hydrolyseerbaar

GTP analoog) of GDP. In tegenstelling tot klassieke G-eiwitten, schakelen Roco eiwitten

dus niet tussen een actieve (GTP) en inactieve (GDP) conformatie, maar is de cyclus zelf

de actieve vorm die de kinase activiteit verhoogd. Met andere woorden; LRRK2 moet GTP

verbruiken om tot maximale kinase activiteit te komen.

In hoofdstuk 5 laten we drie verschillende kristalstructuren van de Mb RocCOR-

tandem dimeer met atomaire resolutie zien. Consistent met de eerdere bevindingen wijst

dit nogmaals op het verschil met de klassieke kleine GTPases. Bovendien hebben we van

deze structuren geleerd dat de drie subdomeinen (Roc, COR-A en COR-B) meerdere

conformaties ten opzichte van elkaar kunnen verwerven. Ook lijkt het duidelijk dat switch

II en de RocCOR interface een belangrijke rol in het activatie mechanisme en de functie

van het RocCOR domein tandem spelen.

In dit proefschrift hebben wij een gecombineerd inzicht kunnen geven in de

kinetische, structurele en biochemische eigenschappen van de G-eiwit cyclus van LRRK2

en Roco eiwitten. Dit heeft niet alleen geleid tot een nieuw model voor het

activeringsmechanisme (hoofdstuk 6 + 7), maar ook belangrijke nieuwe vragen aan het

licht gesteld. Hoe kunnen conformatie veranderingen in de RocCOR tandem leiden tot een

verhoogde kinase activiteit? Welke co-regulatoren beïnvloeden de GTPase activiteit?

Welke rol speelt membraan-binding van LRRK2? Ondanks de belangrijke vooruitgang

geboekt met de productie en isolatie van het humane LRRK2 eiwit, is het panel van de nu

beschikbare bacteriële Roco-eiwitten een waardevolle toevoeging en kan het, zoals in dit

proefschrift aangetoond, een belangrijke rol spelen in het beantwoorden van deze vragen.

159

Acknowledgements

Finally acknowledgements… – Wow, this really is now the last part of my thesis and

actually one of the most difficult for me. I have met so many extraordinary people along

this rather long journey that is my PhD. I am truly thankful to all of you and I try to

appreciate everyone. But I also try to keep it short nonetheless ;)

So, first sentence- first problem: Who should I start with?

I don’t have an answer to that so I just start….

Peter, my first supervisor: You gave me the opportunity to do my PhD in your department,

you were always super nice and kind and you always added an interesting point of view to

all discussions, not only scientifically but also in personal conversations. Also, you are a

great cook! Thank you for everything!

Fred, you gave me the opportunity to do a lot of my work in Dortmund, I cannot thank you

enough for that! You helped whenever needed with all of your knowledge and wisdom.

You are a great boss and an exceptional scientist. It has been an honour to be part of your

group. I think it is very rare to be able to work not only in such an inspiring, efficient and

highly educated environment but also getting along so well with everyone and enjoying

what you are doing. Especially you, Fred, have been a shining example of a passionate

scientist in every aspect. I have learned so much and I will always remember this time

fondly. Thank you so much!

Arjan, my main supervisor: I cannot even attempt to list all the things that you did for me.

Thank you for all the discussions, the constant support, the fact that you have always been

reachable and you are always very positive and supportive about almost everything. Even

when you were traveling, which was quite a lot recently, you were answering my mails

almost immediately and always helping, not only with scientific questions but also

bureaucracy etc. Also, I have to thank you for giving me the opportunity to do my PhD the

way I did, with the close collaboration with the MPI. I think this is far from “normal” to

allow such an arrangement.

160

My dear colleagues in the Netherlands, Ina, my paranymph, Ineke, Maarten, Franz,

Richard, Laura, Marion, Panos, Dominika, Ahmed, Janet but also former colleagues Bernd,

Liu, Rama, Kasia and Ankita. Thank you so much for your warm welcomes. It is a shame

that I didn’t spendt more time with you. Although I was very happy to be able to do most

of my work in Dortmund, I am sad that I didn’t see you more often. Thank you for always

seeing me as a full member of the group.

Ina, special thanks to you for your help and support with organizing everything! You are

such a helpful and happy person, laughing a lot! I am sure you’ll be in my place very soon,

becoming a Dr.

My dear (former) colleagues at the MPI in Dortmund, Steffi my paranymph, Caro, Patricia,

Jana, Eyad, Eldar, Mandy, Rita, Katja, Björn, Mamta, Kim, Shehab, Ben, Denise and

Bernd (again, because you were also part of this group ;) ). Thank you for all your support,

help and fruitful discussions during Pizza seminars and other occasions. It’s been so great

not only working with you in this group but also the lab outings and other “social events”. I

could write so much about what I owe each of you and what I learned from you, but this

would probably fill another book, so I keep it short, I hope you don’t mind: You have been

the people who made this “inspiring, efficient and highly educated environment” that I

mentioned earlier in which working was not only working but meeting friends and having

fun. Thank you for everything!!!

Also, big thanks to our Hiwis Simon, Lars, Luki, Susanne, Janina, Sven and Pascal for

your great help in the lab. And also thank you Nadia and Sibel.

Only one short extra sentence to you Steffi: I am so glad that we both were the last students

in Freds group and that we had each other. It was a great time and I will miss you! Thank

you for everything and for being my paranymph!

I would also like to thank the Xray community at the MPI especially Ingrid Vetter, Georg

Holtermann and Raphael for their support concerning crystallography. Also I would like to

thank Eckard Hofmann from the Ruhr-University for his support and teaching not only at

the SLS.

I also would like to thank some colleagues from other groups namely Matias’ and Heinz’

group for giving me the opportunity to finish my research when they started their groups.

Also thank you Diana, Petra (Geue) and Petra (Neumann), Neha, Martin, Sheila and

161

Glyxia for the great atmosphere, support and nice discussions back in the BMZ. Arsen,

thank you for all your help and support concerning the AUC etc.!

Also I am very grateful to the numerous collaboration partners that I was encountering

during my PhD. I have to thank especially Wim and the members of his group, Lina, Egon

and Margaux for your extremely professional and fruitful collaboration. Wim, I have

learned a lot from you, thank you very much!

I would also like to thank Johannes, Giam, Johann and Katharina for your collaboration

and the very fruitful discussions. Also I owe a big thanks to Jeni Lauer, thank you for your

help on the HDX experiments.

Last but surely not least, there are probably a million other people that I met along the way

and that helped me in one way or the other. There was the introduction course in the very

beginning of my PhD where I met other students from very different fields, there was the

PhD day and the yearly GBB Symposia, numerous conferences, the Klausenhof meeting

from the MPI, the summer school in Greece , the X-ray workshop in South America and

CCP4 study weekends only to name a few. I am grateful to everyone who made this

possible and to everyone who participated. Every event was a great time and I wouldn’t

want to miss it! Also I owe a lot to the scouts, friends and family. Thank you for your

support, for all the great experiences and for making me who I am.

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Chapter 7