University of Groningen
Structural and biochemical characterization of Roco proteins Terheyden, Susanne
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Structural and Biochemical Characterization of Roco Proteins
PhD Thesis
Susanne Terheyden
The research described in this thesis was carried out at the Department of Cell Biochemistry of the Graduate School of Science and Engineering (GSSE) of the University of Groningen and at the Max Planck Institute of Molecular Physiology in Dortmund. This research was funded by the Michael J. Fox Foundation for Parkinson research (MJFF) and the GSSE.
ISBN: 978-94-034-0475-2
© Copyright 2018 Susanne Terheyden
All rights reserved. No part of this thesis can be reproduced, stored in a retrieval system or transmitted in any form of means, without prior written permission of the author and the publisher holding the copyright of the articles.
Cover design: Susanne Terheyden
Printed by: Ipskamp Drukkers, Enschede
Structural and Biochemical Characterization of Roco
Proteins
PhD thesis
to obtain the degree of PhD at the University Groningen 0n the authority of the Rector Magnificus Prof. E. Sterken
and in accordance with
the decisions by the College of Deans.
This thesis will be defended in public on Friday, 25th of May 2018 at 16:15 hours
by
Susanne Terheyden born on the 11th of April 1988 in Recklinghausen, Germany
Supervisor
Prof. dr. P.J.M. van Haastert
Co-supervisor Dr. A. Kortholt
Em. prof. dr. A. Wittinghofer
Assessment Committee Prof. dr. F.W. Herberg Prof. dr. B. Poolman Prof. dr. D.B. Janssen
Contents
Chapter 1
Introduction: The unconventional G-protein cycle of LRRK2 and Roco proteins Aims of the Thesis
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Chapter 2
Revisiting the Roco G-protein cycle 17
Chapter 3
A homologue of the Parkinson’s disease-associated protein LRRK2 undergoes a
monomer-dimer transition during GTP turnover 29
Chapter 4
Biochemical and kinetic properties of the complex Roco G-protein cycle 61 Chapter 5
Structural insights into the Roco G-protein cycle 89
Chapter 6
Regulation of LRRK2 activation, localization and dimerization 119 Chapter 7
Summary and Discussion 149
Nederlandse Samenvatting 158
Acknowledgements 160
Chapter 1
Introduction
The unconventional G-protein cycle of LRRK2 and Roco proteins
Susanne Terheyden, Laura M. Nederveen-Schippers and Arjan Kortholt
This chapter has been published in: Soc. Trans. 2016, 44, 1611–1616.
All authors contributed to the writing of the paper.
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Aim of the Thesis
Roco proteins, including LRRK2, in general are very complex proteins in numerous different aspects. Not only have they been linked to nearly every process within the cell, but also their domain architecture and self-regulation is complicated and not yet completely understood. It gave the impression of a large puzzle where now and then pieces were discovered but they didn’t seem to easily fit to each other yet. This thesis aims to contribute a piece of the puzzle by shedding light on the activation mechanism of the Roco protein family’s name giving feature, the RocCOR domain tandem. Since several of the PD mutations are located in these two domains, it seems clear that the RocCOR domain tandem has important functions in LRRK2 and Roco proteins. Independent of each other, several groups have presented indication that the Roc domain is not a classical small G-protein but might act differently in Roco proteins, but also that the Roc domain plays a central role in LRRK2 activation and PD pathogenesis. Therefore the aim of my thesis is to investigate and biochemically characterize the RocCOR domain tandem and thereby contribute to the understanding of Roco proteins, especially LRRK2 and its malfunction in PD.
The second chapter describes the structure and biochemical features of the RocCOR domain tandem lacking the C-terminal dimerization domain. In the third chapter we discovered that the Chlorobium tepidum (Ct) Roco protein has the ability to monomerize upon GTP binding which was actually very surprising and at first sight opposed to our initial hypothesis, that Roco proteins belong to the group of GADs that dimerize in the presence of GTP. In the fourth chapter we set out to biochemically characterize this Ct Roco protein and several other prokaryotic Roco proteins thoroughly to further elucidate the kinetic behaviour of the hydrolysis reaction. Chapter 5 presents the structures of the Methanosarcina barkeri (Mb) RocCOR tandem bound to different nucleotides. Finally chapter 6 summarizes all these findings in the context of LRRK2, puts some puzzle pieces together to form the bigger picture.
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Chapter 2
Revisiting the Roco G-protein cycle
Susanne Terheyden*, Franz Y. Ho*, Bernd K. Gilsbach*, Alfred Wittinghofer and Arjan Kortholt
*equal contribution
This chapter has been published in: Biochem. J. 2015, 465, 139–147.
ST and BG performed all biochemical and structural experiments with the bacterial proteins and FH with human LRRK2. AW and AK designed the experiments and all authors contributed to the writing of the paper.
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Supplementary Information
Figure S1. Sequence alignment. Homo sapiens (Hs) LRRK2 (Swiss: Q5S007), Chlorobium tepidum (Ct) Roco (Swiss: Q8KC98) and Methanosarcina barkeri (Mb) Roco2 (Swiss:
Q46A62) sequences were aligned using the PRALINE sequence alignment server [1] and GeneDoc (http://www.psc.edu/biomed/genedoc). Black and grey background represents the sequence identity in three or two sequences, respectively. Structural elements of the G- domain (lines), construct boundaries (horizontal arrows), and important residues (vertical arrows) are indicated above the alignment.
1 Simossis, V. A. and Heringa, J. (2005) PRALINE: a multiple sequence alignment toolbox that integrates homology-extended and secondary structure information. Nucleic Acids Res. 33, W28994.
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Chapter 3
A homologue of the Parkinson’s disease-associated protein LRRK2 undergoes a monomer- dimer transition during GTP turnover
Egon Deyaert, Lina Wauters, Giambattista Guaitoli, Albert Konijnenberg, Margaux Leemans, Susanne Terheyden, Arsen Petrovic, Rodrigo Gallardo, Henderikus Pots, Peter J.M. Van Haastert, Frank Sobott, Johannes Gloeckner, Rouslan Efremov, Arjan Kortholt, Wim Versées
This chapter has been published in: Nat. Commun. 2017, 8, 1008.
ED, LW, PV, AP, RE, JG, FS, AK and WV designed the experiments. ED, GG, ML, HP and ST purified the proteins. LW and RE performed EM analysis. ED and RG performed the SEC-MALS analysis. ED and WV performed the SEC-SAXS analysis. ST, AP and ED performed the AUC analysis. AK and ED performed the native MS analysis. ED performed the kinetic and FRET experiments. ED and WV wrote the manuscript. All the authors read and edited the manuscript.
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Supplementary Information
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Chapter 4
Biochemical and kinetic properties of the complex Roco G-protein cycle
Lina Wauters*, Susanne Terheyden*, Bernd K. Gilsbach, Margaux Leemans, Panagiotis A.
Athanasopoulos, Giambattista Guaitoli, Alfred Wittinghofer, Christian Johannes Gloeckner, Wim Versées, Arjan Kortholt
*equal contribution
This chapter has been submitted.
LW and ST performed, with assistance of ML and PA, the biochemical characterization of the prokaryotic Roco proteins. BKG and GG did purify and generated the biochemical data for human LRRK2. LW, ST, AW, WV and AK analyzed results. All authors designed research and contributed to writing of the manuscript.
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Biochemical and kinetic properties of the complex Roco G-protein cycle
Lina Wauters1,2,3*, Susanne Terheyden1,4*, Bernd K. Gilsbach5, Margaux Leemans2,3, Panagiotis S. Athanasopoulos1, Giambattista Guaitoli5, Alfred Wittinghofer4, Christian
Johannes Gloeckner5,6, Wim Versées2,3,■, Arjan Kortholt1, ■
1Department of Cell Biochemistry, University of Groningen, Groningen 9747 AG, The Netherlands
2VIB-VUB Center for Structural Biology, Pleinlaan 2, 1050 Brussels, Belgium
3Structural Biology Brussels, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium
4Structural Biology Group, Max-Planck Institute of Molecular Physiology, 44227 Dortmund, Germany
5German Center for Neurodegenerative Diseases (DZNE), Otfried-Müller-Str. 23, 72076 Tübingen, Germany
6University of Tübingen, Institute for Ophthalmic Research, Center for Ophthalmology, 72076 Tübingen, Germany
*equal contribution
■corresponding authors
Abstract
The Roco protein family has come into focus after mutations in the gene coding for the human Roco protein LRRK2 (Leucine-rich repeat kinase 2) were discovered to be the most common genetic cause of familial forms of late onset Parkinson’s disease. Roco proteins are characterized by a Roc domain (Ras-like G-domain) responsible for GTP binding and hydrolysis, followed by a COR (C-terminal of Roc) dimerization device. The regulation and function of the RocCOR domain tandem is still not completely understood. To fully biochemically characterize Roco proteins, we performed a systematic survey of the kinetic properties of a set of Roco protein family members, including LRRK2. Together, our results show that Roco proteins have a unique G-protein cycle. Our results confirm that Roco proteins have a low nucleotide affinity in the micromolar range and thus do not strictly depend on G-nucleotide exchange factors (GEFs). Measurement of multiple and single turnover reactions shows that neither Pi release nor GDP release are rate limiting, while this is the case for the GAP-mediated GTPase of some small G proteins like Ras or Rap and for most other high affinity Ras-like proteins, respectively. Strikingly, the KM values of the reactions are in the range of the physiological submillimolar GTP concentration, suggesting that LRRK2 activity and function might be regulated by the cellular level of GTP.
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Abbreviation list
LRRK2 Leucine-rich repeat kinase 2
PD Parkinson’s disease
GTP Guanosine triphosphate
GDP Guanosine diphosphate
Pi free (inorganic) Phosphate
IPTG Isopropyl β-D-1-thiogalactopyranoside
DTT dithiotreitol
β-Met β-Mercaptoethanol
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Introduction
Roco proteins were originally described in 2003, as a novel group of the Ras superfamily (1).
They are large multidomain proteins characterized by a tandem organization of a Ras-like G- domain (Roc, Ras of complex proteins) and a COR (C-terminal of Roc) dimerization device.
The Roco protein family came into the focus of research when mutations in LRRK2, a human Roco protein, were shown to segregate with Parkinson’s Disease (PD) (2,3). PD-related mutations and risk factors are mostly found in the catalytic core of the protein, formed by the RocCOR supra-domain followed by a kinase domain (4). So far, it remains to be determined if the kinase, the RocCOR tandem or both regulate the output signal of LRRK2 (5–7).
Nevertheless, the RocCOR and kinase domains are both essential for proper LRRK2 functioning (3). Disruption of nucleotide binding results in reduced kinase activity of LRRK2 (8–11), suggesting that the kinase activity of LRRK2 is dependent on the Roc domain.
Importantly, the most prevalent PD mutations result in an increased LRRK2 kinase activity and/or decreased GTPase activity (6,9,12–18). Despite the important role of the RocCOR tandem, its regulation is still not completely understood.
The Roc domain shows high similarity to the family of small G-proteins (1). G-proteins are Guanine nucleotide-binding proteins that typically switch between an active GTP- and inactive GDP-bound state (19,20). G-proteins can occasionally also function as genuine GTPases that use GTP hydrolysis directly to energetically drive chemical reactions or mechanical work in the cell (21). In exceptional cases, G-proteins can act as a sensor of the GTP status in the cell (22). Depending on their mode of action, G-proteins can be divided into functionally different classes. Up to date, there is still an ongoing debate whether the Roc domain of Roco proteins functions as a conventional G-protein or as a GAD (G-proteins activated by nucleotide-dependent dimerization) (23). Both conventional G-proteins and GADs, function as molecular switches, however their cycle is regulated in a completely different way. A number of studies from our and other labs have found that some Roco proteins, including LRRK2, have a relatively low affinity for nucleotides, as well as a fast dissociation of nucleotides (11,24–27). This suggests that Roco proteins, like GADs and in contrast to conventional small G-proteins, do not require Guanine nucleotide Exchange Factors (GEFs) for their activation. Moreover, some Roco proteins, including LRRK2, have been shown to function as dimers, also favoring a GAD-like GTPase mechanism (24,28).
However, our labs have recently shown that the Roco protein from Chlorobium tepidum
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undergoes a dimer-monomer transition upon GTP binding and cycles between a monomeric and dimeric form during GTP turnover, thus challenging a simple GAD model (29).
Here we performed a systematic and detailed survey of the kinetic properties of several prokaryotic Roco proteins and LRRK2. Together, our data show that Roco proteins have a unique G-protein cycle and suggest that the GTPase reaction consist of multiple steps and can be influenced by changes in cellular GTP levels.
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Results and Discussion
Roco proteins have G-nucleotide affinities in the low micromolar range
In this study, the biochemical properties of the Roco proteins were investigated using five prokaryotic Roco protein family members from Methanosarcina barkeri (MbRoco1 and MbRoco2), Spirosoma linguale (SlRoco), Nostoc punctiforme (NpRoco), and Chlorobium tepidum (CtRoco), and full-length human LRRK2 (HsLRRK2). All of these proteins contain a Leucine-Rich repeat (LRR), a Roc and a COR domain. In the case of MbRoco2, we also used the RocCOR tandem for comparison. The Roc domain, especially the catalytically important P-loop, switch I and switch II motif, are highly conserved from prokaryotic to eukaryotic Roco proteins (Fig. 1). The prokaryotic Roco proteins were expressed in E.coli, while the full-length HsLRRK2 protein was purified from HEK293T cells (Table S1 and Fig. S1).
Figure 1: Domain topology and alignment of Roco proteins used in this study. Schematic domain topology of LRRK2 and prokaryotic Roco proteins. PD-associated mutations and risk factors are indicated. Lower panel: Sequence alignment (clustalW) of parts of the Roc and COR domain highlighting PD mutations and G-domain elements.
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Conventional small G-proteins like Ras, Rab and Arf have very high nucleotide affinities which results in very low dissociation rates (30). Therefore, this group of proteins requires the help of GEFs that reduce the nucleotide affinity from the picomolar to the micromolar range to enable the protein to exchange the nucleotide and to switch from the inactive GDP state to the active GTP state (19,31). In contrast, GADs, including MnmE, dynamin and septins (32–34), have low nucleotide affinities and therefore do not depend on GEFs (35).
To determine the binding affinities of the Roco proteins for GDP and GTPyS (a non- hydrolysable GTP-analogue under the conditions used), we performed fluorescence anisotropy equilibrium titration experiments. The mant-labeled derivatives of GDP or GTPyS were titrated to the nucleotide-free Roco proteins and binding was monitored as the increase in fluorescence anisotropy signal (Fig. 2). Since LRRK2 is unstable in the absence of nucleotide, we could not determine the nucleotide affinity for this protein. However, all other Roco proteins, except CtRoco, have binding affinities (KD) for GDP and GTPγS in the low micromolar range with a consistently higher affinity for GTPγS compared to GDP (Table 1, Fig. 2 and S2). CtRoco also has a low affinity for GDP, however it has a much higher affinity for GTPyS. Indeed, even when using a very low mant-GTPγS concentration (0.2 μM), the titration curve is clearly biphasic indicating that the dissociation constant is well below this concentration. Therefore, the exact value for the binding constant is indeterminable using this technique. Despite the variation in the affinity for GTPyS, all Roco proteins have an affinity for GDP in the low micromolar range, with fast nucleotide dissociation rates (koff, table 1), thus making GEFs obsolete.
Roco proteins have a low to moderate GTP hydrolysis rate
Although conventional small G-proteins are also called GTPases, their catalytic machinery to perform hydrolysis is incomplete. For this reason, they have co-evolved with GTPase- activating proteins (GAPs) that complete the catalytic machinery and in this way stimulate the intrinsic slow GTP hydrolysis rate (31,36). GADs do not require GAPs for hydrolysis.
After binding of GTP, the G-domains of these proteins dimerize to complement each other’s active site and in this way activating GTP hydrolysis (35,37). We determined the steady state kinetics of the GTPase reaction of the Roco proteins using a reversed phase HPLC assay where the different G-nucleotides are separated by their retention time (Fig. 3;
Fig. S3). The kcat value for all Roco proteins falls between 0.06 min-1 and 0.5 min-1; in other words it takes one Roco protein between 2 to 10 minutes to convert one GTP molecule to GDP. This is faster than the intrinsic GTP hydrolysis of most classical small
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G-proteins, but much slower than GAP-catalyzed GTP hydrolysis. For example, the intrinsic hydrolysis rate of Ras is 0.03 min-1 at 37°C (38) and the GAP stimulated rate is 300-600 min-1 at 25°C (39). Moreover, the GTP hydrolysis rate of Roco proteins is well in line with the intrinsic hydrolysis rate of GADs like MnmE. The basal GTP hydrolysis rate of MnmE is 0.33 min-1 (40). However, when bound to its interaction partner MnmG or upon stimulation by K+-ions, the hydrolysis rate of the protein increases 5-fold (1.7 min-1) to 20-fold (7.8 min-1). This could suggest that also for Roco proteins, the GTP hydrolysis can be triggered by direct or indirect interaction with small ligands or other proteins.
Figure 2: Equilibrium binding titrations showing binding of MbRoco2 and CtRoco to mant-GDP and mant-GTPγS. The binding isotherms were obtained by following binding of protein at decreasing concentrations to a fixed concentration of mant-nucleotide using the fluorescence anisotropy signal for a) MbRoco2 binding to GDP, b) CtRoco binding to GDP, c) MbRoco2 binding to GTPγS, d) CtRoco binding to GTPγS. Each data point is the average (± s.e.m.) of three independent anisotropy measurements from at least two independent biological samples. The resulting nucleotide affinities (± s.e.) are given.
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Table 1: Summary of binding and kinetic parameters of different Roco proteins.
KD GDP
[µM] KD GTPγs
[µM] KM
[µM] kcat
[min-1] koff GDP
[min-1] koff GTPγs [min-1] MbRoco1 48
± 9 (n=3)
± 0.5 1.7 (n=3)
± 134 448 (n=3)
0.13
± 0.01 (n=4)
14.20
± 0.02 (n=1)
14.24
± 0.03 (n=1)
MbRoco2 f.l.
± 3 9 (n=3)
± 0.6 2.9 (n=3)
± 74 743 (n=3)
0.068
± 0.002
(n=3)
12.78
± 0.07 (n=2)
12.60
± 0.05 (n=2)
MbRoco2
RocCOR 1.8c 0.18a
± 0.08 (n=3)
± 129 353 (n=3)
0.063
± 0.006
(n=3)
0.33c 10.35
± 0.03 (n=1)
SlRoco 55
± 14 (n=3)
± 12 51 (n=3)
1097
± 605 (n=3)
0.30
± 0.06 (n=3) - -
NpRoco 23
± 3 (n=3)
± 0.6 3.0 (n=3)
± 41 354 (n=3)
0.109
± 0.004
(n=3)
± 6 230 (n=1)
14.8
± 0.1 (n=3)
CtRoco 21
± 3 (n=3)
<0.5a
(n=3)
<15b
(n=4)
0.066
± 0.002
(n=3)
± 2 106 (n=3)
0.479
± 6.10-4
(n=3)
HsLRRK
2 - - 400
± 116 (n=4)
0.517
± 0.047
(n=4)
- -
This table reports all parameters obtained by fluorescence anisotropy equilibrium titrations (KD), Michaelis-Menten kinetics (kcat, KM) and stopped-flow fluorescence polarization experiments (koff GDP/GTPγS). n represents the number of independent repetitions. For n=1, the value (± s.d.) is given. For n>1, the parameter (± s.e.) is indicated.
a Value of the constant ligand concentration (0.2 µM) used in the titration is higher than the fitted value of KD. Care should thus be taken since this could give rise to an overestimation of the reported KD. In extreme cases an upper limit of the KD is given.b KM is lower than the lowest measurable GTP concentration. c Value previously published in Terheyden et al., 2015 (26).
Figure 3: Steady state (Michaelis-Menten) kinetics of GTP hydrolysis by MbRoco2 (a) and CtRoco (b). Every data point is the average (± s.e.m.) of at least three independent measurements and two biological replicates. By fitting to the Michaelis-Menten equation, the Michaelis-Menten constant (± s.e.) (KM) and turnover number (± s.e.) (kcat) were determined for MbRoco2 (a) and CtRoco (b). For CtRoco, the KM value was lower than the lowest GTP concentration measured so a lower limit of 15 μM is given.
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Enzyme turnover is not limited by product release
The overall kinetics of the GTPase reaction can be governed by the chemical conversion of GTP to GDP and the associated conformational changes, the GDP release or the Pi release.
For the GAP-mediated GTP hydrolysis by Ras and Rap, it has been shown that Pi release together with the associated conformational changes is the rate limiting step (41,42). In contrast for MnmE, a member of the GAD family, dissociation of the G- domain dimer after GTP hydrolysis has been proposed to be rate limiting under certain circumstances (43). We first used stopped-flow fluorescence anisotropy measurements to determine the rate limiting step of the Roco GTPase reaction (Fig. 4, Fig. S4, table 1). In-line with the observed low affinity for GDP, the GDP off-rates of all Roco proteins are in the range of 12 min-1 to 230 min-1. Since the GDP off rates are much faster than the kcat values (0.063 min-1 to 0.3 min-1), GDP dissociation is not rate limiting in the full enzymatic cycle (see table 1).
Figure 4: Rate of GDP and GTPyS dissociation (koff) from CtRoco and MbRoco2.
Representative traces showing the dissociation of mant-GDP and mant-GTPS from CtRoco and MbRoco2 followed by stopped-flow fluorescence polarisation upon mixing mant-nucleotide-bound protein with an excess of unlabeled nucleotides. The koff rates (±
s.e.) depicted result from several repetitions (see table 1). a) Dissociation of GDP from MbRoco2, b) Dissociation of GDP from CtRoco, c) Dissociation of GTPyS from MbRoco2, d) Dissociation of GTPyS from CtRoco.
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To determine if the actual hydrolysis step or Pi release is rate limiting, we measured the single turnover GTP hydrolysis rate (kchem). Since these measurements require a high amount of protein, we performed these experiments with the highly expressed CtRoco protein. The single turnover rate at saturating enzyme concentration (kchem) is 0.06 ± 0.01 min-1, (Fig. S5), which is in the same range as our observed kcat value. Since a kchem
value reflects the rate of all on-enzyme steps until, but not including, product (GDP and Pi) release, this confirms that neither nucleotide nor Pi release are rate limiting.
KM values for the GTPase reaction coincide with physiological GTP concentrations Strikingly, the KM values for the GTPase reaction are much higher than the KD values for GTPγS binding (Table 1). Interestingly, except for the CtRoco protein, the KM values of all Roco proteins are in the high micromolar range, coinciding with physiological GTP concentrations in the cell (44). Probably due to the high affinity for GTP, the KM of CtRoco is in the lower micromolar range at 20°C. However, it should be noted that the corresponding organism, Chlorobium tepidum, is the only thermophile in the group of proteins described here and known to be able to survive at very low levels of ATP (low light) and thus at presumably low GTP levels (45). Since, C. tepidum has an optimum growth temperature of 48-50°C, we also determined the multiple turnover Michaelis- Menten kinetics experiment at higher temperatures (Fig. S6). Whereas at 20°C, the KM was too low to be determined (see above), at 40°C a KM of 18 ± 3 µM could be determined (Fig. S6). This indeed confirms an increase of KM of the protein at higher temperatures and suggests that, at physiological temperatures, the KM might be in a similar range as the KM values of the other Roco proteins, presented here.
Full-length LRRK2 (Fig. S3) has a KM of 400 µM, which is in a similar range as previously published for a Roc domain (553 µM) (27) and a RocCOR-kinase fragment (343 µM) (46). In addition, this value is also similar to those of the bacterial Roco proteins, determined here (see above). When the KM lies in the range of the substrate concentration, the catalytic activity is directly and sensitively dependent on changes in the substrate concentration and can be tuned according to the needs of the host. Our data might thus suggest that the activity of Roco proteins can be influenced by the cellular levels of GTP and thus could act as a GTP sensor. The first reported example of such a sensor is a phosphoinositide kinase (22). PI5P4Kβ detects the GTP levels in the cells and converts them into lipid second messenger.
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Kinetic model for the unique Roco G-protein cycle
This work, for the first time, provides detailed kinetic insight into the complex Roco G- protein cycle (Fig. 5). Our equilibrium binding studies demonstrated that all Roco proteins have a low affinity for GDP with KD values in the micromolar range, associated with fast GDP dissociation rates, in common. This confirms previous observations that Roco proteins do not require GEFs for the exchange of nucleotides (24,26,47). Because of the much higher physiological concentration of GTP compared to GDP and the rather slow GTP hydrolysis, this also implies that, within the cell, Roco proteins are primarily in the GTP-bound state, therefore not acting as classical molecular switches. Roco proteins have a unique and complex hydrolysis mechanism. Michaelis-Menten kinetics show that the KM
values are much higher than the nucleotide affinities, indicating that the GTPase mechanism consists of multiple steps. Comparison of single (kchem; function of k2, k-2 in Fig. 5) and multiple (kcat; function of k2, k-2, k3, k-3, kon,GDP, koff,GDP in Fig. 5) turnover kinetics, in combination with the GDP release rates (koff), revealed that GTP hydrolysis itself or conformational changes occurring during or after nucleotide binding (represented by k2, k-2) but prior to product release, are rate limiting. Roco proteins have moderate intrinsic GTP hydrolysis rates, suggesting that the GTPase might require co-factors.
Indeed, for LRRK2, it has been reported that binding of effectors or GTPase co-regulators, including ARHGEF7 (48), ArfGAP1 and RGS2 (49–51) modulates the hydrolysis reaction.
In addition, the GTPase activity might be triggered by localization to intracellular membranes or vesicles and/or by dimerization (29,52). This is supported by the fact that LRRK2, presumably due to higher local concentration, dimerizes at the membrane, where it has a higher kinase activity (53). Importantly, we found that Roco proteins have a KM
value in the range of the physiological GTP concentration within cells, suggesting that the GTPase activity and function of Roco proteins might be directly and sensitively dependent on the cellular GTP concentration.
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Figure 5: Model of the kinetic mechanism of Roco proteins. GTP binding is followed by rate-limiting GTP hydrolysis and the associated conformational changes. Product (Pi, GDP) release is not rate-limiting.
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Materials and Methods:
Purification of prokaryotic Roco proteins and LRRK2
The purification of full-length LRRK2 has been previously described (54). Detailed information on expression vectors, cells, temperatures, as well as purification steps and the final purification buffers can be found in Table S1. As a final purification step, size exclusion chromatography was performed. Purified proteins were analysed by SDS-PAGE, and the protein concentration was determined using the absorption at 280 nm (Nanodrop 2000, Thermo Scientific). Before starting the experiments, the nucleotide load of each protein batch was determined via reversed phase chromatography.
Equilibrium titrations
Nucleotide-free protein was used in an equilibrium titration experiment to measure the affinity for 3’-O-N-methylanthraniloyl (mant)-labeled GDP and GTPyS nucleotides.
Buffer containing 1 µM mant-nucleotide was titrated stepwise in a cuvette containing protein and 1 µM mant-nucleotide at 20 °C. In this way, the protein was stepwise diluted, while keeping the mant-nucleotide concentration constant, until the signal of free mant- nucleotide was reached. Fluorescence anisotropy was measured using a FluoroMax-4 fluorimeter (Horiba Scientific, excitation: 360 nm, emission: 450 nm). The signal was recorded and averaged over at least 5 min. As a buffer, the final purification buffer (Table S1) was used, without reducing agent but including at least 5 mM MgCl2. The data was then plotted using Prism 7 (Graphpad software). Each data point is the average (± s.e.m.) of three independent measurements. The dissociation constant (KD) (± s.e.) was then calculated by fitting a quadratic equation to the data.
Steady-state kinetic measurements of GTP hydrolysis
In order to measure the Michaelis-Menten constant (KM) and turnover number (kcat) of the GTP hydrolysis reactions, a HPLC-based assay was used as previously described (55).
Briefly, 1 µM of protein (0.1 µM for LRRK2) was incubated with the indicated GTP concentrations at 20°C. For CtRoco and NpLRRP, 50 μl samples were taken at several time points and boiled for 5 min at 95°C to stop the reaction. After centrifugation (10 min, max. speed), 35 μl supernatant was added to 35 μl HPLC-buffer containing 100 mM KH2PO4 pH 6.4, 10 mM tetrabutyl NH4Br and 7.5% acetonitrile, and finally 50 μl of this sample was injected on a reversed phase C18 column connected to a e2695 HPLC system (Waters) to separate GTP from GDP. The area under the curve for GDP was converted to
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concentration using a standard curve. For the other proteins the sample volume was 5 μl and samples were directly injected on a reversed phase C18 column using an Ultimate 3000 HPLC system (Thermo Scientific) in HPLC-buffer containing 50 mM KH2PO4/K2HPO4 pH 6.4, 10 mM tetrabutyl NH4Br and 15% acetonitrile. Subsequently, samples were analysed using the HPLC integrator (Chromeleon 7.2, Thermo Scientific).
Initial rates of GDP production were plotted against the GTP concentration using Prism 7 (Graphpad software). Each data point is the average (± s.e.m.) of three independent measurements. The Michaelis-Menten equation was fitted to determine KM (± s.e) and kcat (± s.e). As reaction buffer, the final purification buffer (Table S1) was used, without reducing agent but containing at least 5 mM MgCl2.
Nucleotide binding kinetics
For the measurement of nucleotide binding kinetics, the stopped-flow apparatus SX18MV (Applied photophysics) equipped with a 20 µl cell (50 µl per shot) was used in fluorescence polarization mode. Fluorescence polarization was recorded at 20°C using an excitation wavelength of 360 nm and an emission filter of 420 nm. For determination of the dissociation rate (koff), 5 µM of mant-nucleotide bound to 10 µM of protein was rapidly mixed with 1 mM of unlabelled nucleotide. The koff (± s.e) was determined using a single exponential fit in Prism 7 (Graphpad software). As a buffer, the final purification buffer was used (Table S1), without reducing agent but containing at least 5 mM MgCl2.
Single turnover kinetics of GTP hydrolysis
To determine the single turnover reaction rate of GTP hydrolysis (kchem), 5 μM GTP was added to increasing protein concentrations. As a buffer, the final purification buffer was used (Table S1), without reducing agent but containing at least 5 mM MgCl2. At several time points, samples were taken and GTP was separated from GDP using reversed phase chromatography as described above. Using a GDP standard, the area under the curve of each peak was used to determine the amount of GDP in the sample. The reaction rate at each protein concentration (kobs) was determined by fitting the GDP concentration in function of time on a single exponential using Prism 7 (Graphpad software). The kobs value at a saturating concentration of protein of 30 µM (GTP binding much faster than GTP hydrolysis) corresponds to the single turnover kchem. The measurements were performed in triplicates.
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Acknowledgements
We would like to thank H. Pots for the technical assistance. This research was supported by a VUB/RUG collaboration agreement (OZR2544; L.W.), the Fonds voor Wetenschappelijk Onderzoek (M.L., W.V.), a Strategic Research Program Financing of the VUB (W.V.), The Michael J. Fox Foundation for Parkinson’s Research (A.K., W.V., C.J.G.), iMed (C.J.G) and a NWO-VIDI grant (A.K.).
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Supplementary information
Figure S1: SDS-Page gels showing sample purity of protein constructs. a) MbRoco2 full length, b) MbRoco2 RocCOR, c) MbRoco1, d) SlRoco, e) CtRoco, f) NpRoco and g) HsLRRK2. For samples (a) to (d) a Pierce Unstained Protein MW Marker was used, for samples (e) and (f) a Pageruler Prestained Protein MW Marker and for sample (g) a Fermantas Pageruler Plus MW Marker. The MW’s depicted next to the markers are given in kDa.
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Figure S2: Equilibrium binding titrations of Roco proteins to mant-GDP and mant-GTPγS.
Anisotropy measurements were performed to determine the protein’s affinity for GDP and GTPγS. The binding isotherms were obtained by following binding of decreasing concentrations of protein to a fixed concentration of mant-nucleotide using the fluorescence anisotropy signal. Each data point is the average (± s.e.m.) of three independent anisotropy measurements from at least two different protein stocks. The resulting affinity for the nucleotide (± s.e.) is indicated in the respective figure. a) MbRoco1 binding to GDP; b) MbRoco1 binding to GTPγS; c) NpRoco binding to GDP; d) NpRoco binding to GTPγS; e) SlRoco binding to GDP; f) SlRoco binding to GTPγS; g) MbRoco2 RocCOR binding to GTPγS. The affinity of MbRoco2 RocCOR for GDP was published in Terheyden et al., 2015.
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Figure S3: Steady state (Michaelis-Menten) kinetics of GTP hydrolysis. Every data point is the average (± s.e.m.) of at least three independent measurements from at least two different protein stocks. By fitting to the Michaelis-Menten equation, the Michaelis- Menten constant (± s.e.) (KM) and turnover number (± s.e.) (kcat) were determined. a) MbRoco1, b) NpRoco, c) SlRoco, d) MbRoco2 RocCOR, e) HsLRRK2.
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Figure S4: Rate of GDP and GTPγS disscociation (koff) for Roco proteins. Representative stopped-flow traces showing the dissociation of mant-GDP and mant-GTPγS from Roco proteins. The koff rates depicted result from one or several (± s.e.) repetitions (see table 1).
a) MbRoco1 GDP koff measurement (n=1), b) MbRoco1 GTPγS koff measurement (n=1), c) NpRoco GDP koff measurement (n=1), d) NpRoco GTPγS koff measurement (n=3), e) MbRoco2 RocCOR GTPγS koff measurement (n=1).
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Figure S5: Single turnover kchem measurement for CtRoco. First, single turnover GTP hydrolysis by CtRoco was measured at various CtRoco concentrations (using 5 µM GTP), and traces were fitted to a single exponential to obtain kobs. When using 30 µM protein or more, the kobs value became quasi-independent of the CtRoco concentration indicating that the rate of chemistry (kchem) is reached. With this protein concentration (30 µM), three repetitions were performed resulting in the kchem rate (± s.e.) depicted.
Figure S6: Michaelis-Menten kinetics of the CtRoco protein at 40°C. By fitting to the Michaelis-Menten equation, the Michaelis-Menten constant (± s.e.) (KM) and turnover number (± s.e.) (kcat) were determined.
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