Two helices control the dynamic crosstalk between the catalytic domains of LRRK2

The two major molecular switches in biology, kinases and GTPases, are both contained in the Parkinson’s Disease-related Leucine-rich repeat kinase 2 (LRRK2). Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and Molecular Dynamics (MD) simulations, we generated a comprehensive dynamic allosteric portrait of the C-terminal domains of LRRK2 (LRRK2RCKW). We identified two helices that shield the kinase domain and regulate LRRK2 conformation and function. One docking helix in COR-B (Dk-Helix) tethers the COR-B domain to the αC helix of the kinase domain and faces its Activation Loop, while the C-terminal helix (Ct-Helix) extends from the WD40 domain and interacts with both kinase lobes. The Ct-Helix and the N-terminus of the Dk-Helix create a “cap” that regulates the N-Lobe of the kinase domain. Our analyses reveal allosteric sites for pharmacological intervention and confirm the kinase domain as the central hub for conformational control.


Introduction
Parkinson's Disease (PD), a major neurodegenerative disorder, is characterized by 31 chronic and progressive loss of dopaminergic neurons. Mutations in the PARK8 gene which 32 codes for the Leucine-Rich Repeat Kinase 2 (LRRK2) are the most common cause for genetically 33 driven PD [1]. LRRK2 is a large multi-domain protein that contains an armadillo repeat motif 34 (ARM), ankyrin repeat (ANK), leucine-rich repeat (LRR), ras-of-complex (ROC) GTPase, C- 35 terminal of ROC (COR), protein kinase, and WD40 domains [2]. While crosstalk between kinases 36 and GTPases, the two most important molecular switches in biology, are well-known features in 37 cellular signaling, LRRK2 is one of the few proteins that contains both catalytic domains in the 38 same polypeptide chain [3]. GTP binding to the ROC domain is thought to regulate kinase 39 activity as well as stability and localization [4,5]. Most of the well-known familial mutations are 40 clustered within the ROC, COR and kinase domains; N1473H and R1441C/G/H in the GTPase 41 domain and Y1699C in COR-B lie at the interface between the ROC and COR domains, while 42 G2019S and I2020T are in the highly conserved DFGψ motif within the kinase domain [6,7]. This 43 information collectively suggests that there is considerable crosstalk between the two catalytic 44 domains of LRRK2, but can we capture this crosstalk? 45 We had previously shown that the kinase domain of LRRK2 is a highly regulated 46 molecular switch. Its conformation regulates more than just kinase activity and plays a crucial 47 role in the intrinsic regulatory processes that mediate subcellular location and activation of 48 LRRK2 [8]. Recent breakthroughs in obtaining structure information, including the in situ cryo 49 electron tomography (cryo-ET) analysis of LRRK2 polymers associated with microtubules and 4 50 the high resolution cryo electron microscopy (cryo-EM) structure of the catalytic C-terminal 51 domains (LRRK2 RCKW ), have provided invaluable structural templates that enabled us to achieve 52 a mechanistic understanding of LRRK2 [9,10]. Most recently the cryo-EM structure of full length 53 LRRK2 was also solved at high resolution [11]. 54 Here, we combined hydrogen-deuterium exchange mass spectrometry (HDX-MS) and 55 Gaussian Accelerated Molecular Dynamics (GaMD) simulations to gain insight into the dynamic 56 features of LRRK2 RCKW , a construct that includes both the kinase and GTPase domains. To build 57 a comprehensive allosteric and dynamic portrait of LRRK2 RCKW , we first mapped our HDX-MS 58 data onto the LRRK2 RCKW cryo-EM structure which gave us a portrait of the solvent accessibility 59 of each peptide. We also assessed the effect of the type I kinase inhibitor MLi-2 and finally used 60 GaMD simulations to monitor the dynamics of LRRK2 RCKW . 61 The intrinsic dynamic features of LRRK2 RCKW revealed by HDX-MS and GaMD simulations 62 show how the kinase domain is allosterically regulated by its flanking domains. These two 63 techniques allow us to explore the molecular features of domain:domain interfaces and loop 64 dynamics. In this way we identified two distinct motifs that control the kinase domain. These 65 two motifs, the COR-B docking helix (referred to as the Dk-Helix) and the C-terminal helix (Ct-66 Helix), both impact the overall breathing dynamics of LRRK2 RCKW . In addition, we showed how 67 the Activation Segment (AS) of the kinase domain faces the ROC:COR-B interface. This interface 68 is unleashed by several PD mutations that cluster in the kinase domain and at the interface 69 between COR-B and the ROC domain. The AS is disordered in the LRRK2 RCKW cryo-EM structure, 70 and GaMD simulations allowed us to explore this space. In this inactive conformation, the Dk-5 71 Helix is stably anchored onto the αC helix in the N-lobe of the kinase domain, which locks the 72 C helix into an inactive conformation. 73 74

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Global dynamic portrait of LRRK2 RCKW is revealed by HDX-MS and GaMD simulations 76 To identify the solvent exposed regions of LRRK2 RCKW , we mapped the HDX-MS data 77 onto the cryo-EM structure of LRRK2 RCKW [10] (Fig 1a). Our overall HDX-MS coverage of 78 LRRK2 RCKW , which was >98% (S1 less deuterium uptake, which indicates that its core is less dynamic in solution.  The Dk-Helix has charged residues at both its N-and C-termini while the middle part of 178 the Dk-Helix is amphipathic (Fig. 3b). Multiple hydrophobic residues face the core of the COR-B 179 domain making this a very stable interface (Fig. 3c). On the opposite side, multiple charged 180 residues interact strongly with the αC helix of the kinase domain through electrostatic 181 interactions, locking the C helix into an "out" and inactive conformation (Fig. 3c). Typically, in 182 an active kinase the basic residue that lies at the beginning of the αC helix (R1915 in LRRK2) 183 interacts with a phosphate on the A-Loop, which is missing in this structure [14,15]. Our 184 hypothesis is that the N-terminus of the αC helix very likely becomes "unleashed" when the 185 kinase is in an active conformation. 186 The N-terminus of the Dk-Helix interacts with the COR-B-Kinase linker that is more 187 solvent exposed and wraps around the N-lobe of the kinase domain (Fig. 3a). This region serves  195 This loop is solvent exposed and also disordered in the LRRK2 RCKW structure. Our MD 196 simulations show a dominant interaction of the C-terminal residue, E2527, both the side chain 197 and the -carboxyl group, with R1771 and K1772 at the beginning of the Dk-Helix (Fig. 4b).
198 backbone carbonyl of W1791 and helps to "cap" the Dk-Helix (Fig. 4c and d). The C-terminus of 222 the Dk-Helix also faces the A-Loop of the kinase domain, which is likewise disordered in the 223 LRRK2 RCKW cryo-EM structure (Fig. 3a). We know that this is a critical region because exchange The simulations suggest, for example, that the side chain of R1441 can also interact with E1790, 230 a residue that is anchored to R1915 in the C helix of the kinase domain ( Fig. 3c and 4e). This is 231 the residue that would be predicted to interact with the phosphorylation site (P-site) in the A- can also interact with R2523 (Fig. 6b). In addition, the two hydroxyl groups from T2524 and  is closer to the COR-B-Kinase linker (Fig. 6a). When the C-terminal tail is more distant from the 298 N-lobe of the kinase, pT2524 binds to K1772 and R1723 (S6b Fig.). The different networks that 299 can be mediated upon phosphorylation can clearly affect the breathing dynamics of LRRK2 and 300 could in turn affect LRRK2 activity. Also, yet to be resolved is whether 14-3-3 binding would 301 stabilize an active or an inactive dimer.

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Capturing the dynamics of the Activation Loop 303 As a frame of reference for the LRRK2 AS we show the AS of the cAMP-dependent 304 protein kinase (PKA) when the A-Loop is phosphorylated and the kinase is in a fully closed 305 conformation (Fig. 7a). In PKA the AS begins with the DFGψ motif and ends with the APE motif, 306 two of the most highly conserved motifs in the protein kinase superfamily [23]. In between 307 these two motifs are the A-Loop and the P+1 Loop. The APE-F linker that follows the AS, which 308 typically play an important role in docking of substrates and other proteins [13], and should be 309 considered an extended part of the AS. Our HDX-MS results show that the A-Loop and part of 310 the P+1 Loop in the LRRK2 kinase domain are highly dynamic and likely unfolded ( Fig. 2 and S1 16 311 movie), which is consistent with the fact that the A-Loop and most of the AS are not resolved in 312 the LRRK2 RCKW cryo-EM structure (Fig. 7b). The region extending from the APE motif through to 313 the F helix is, however, folded, and overlays well with the corresponding region of PKA. 314 Several key residues in this region face out towards the solvent, with the corresponding 315 residues in PKA serving as a docking site for the regulatory subunits (R) (S7 Fig.). Y2050 is also 316 highly conserved in most kinases and it bridges to the backbone residues of this P-site residue 317 when the active kinase is phosphorylated on its A-Loop[24,25] (S8 Fig.). In the cryo-EM H-I loop. In the LRRK2 RCKW cryo-EM structure these two residues are close but not within 326 hydrogen bonding distance; however, the interaction between E2042 and R2122 is captured 327 frequently in the GaMD simulations (S9 Fig.). 335 Surprisingly, when looking at the inactive cryo-EM structure of full-length LRRK2, which 336 also contains ATP, the AS is mostly ordered except for 3 residues (Fig. 7c). In this structure the 337 P+1 loop is ordered in a way that overlays well with PKA. However, the DFGψ motif region is 338 ordered in a helix that is buttressed up against the N-lobe in contrast to an active conformation 339 where the DFGψ motif would be fused to a beta strand that binds to the C-lobe (S8 Fig.). Based portrait of LRRK2 RCKW . Based on the solvent-shielded and solvent-exposed regions, we defined 354 three rigid bodies, and were able to confirm this domain organization using GaMD simulations 355 (S11 Fig.). opening and closing of the catalytic cleft ( Fig. 8 and S12 Fig.). MLi-2, a type I kinase inhibitor, All author reviews the manuscript.

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Competing Interests statement 493 The authors have no competing interests. 494