Conformation and Dynamics of the Kinase Domain Drive Subcellular Location and Activation of LRRK2.

Abstract

Background: Leucine-Rich Repeat Kinase 2 (LRRK2) is a complex multi-domain protein where LRRK2-mutations are associated with Parkinson´s Disease (PD). To explore how pathogenic PD-mutations hijack the finely tuned activation process of LRRK2, we here used a multi-tiered approach. Methods: First, the spatial and temporal distribution of full-length LRRK2 was investigated by a real-time cell-based assay in the presence and absence of LRRK2-kinase inhibitors. In a 2nd layer we explored the consequences of PD mutations as well as removal of the N-terminal domains employing a construct containing the ROC, Cor, Kinase and WD40 domains (LRRK2RCKW). We focused on the biochemical characterization of LRRK2RCKW variants based on kinase assays using Rab8a or LRRKtide as substrates. Next, we used hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map the solvent accessible regions of LRRK2RCKW in the presence and absence of the LRRK2 inhibitor MLi-2. Finally, Molecular Dynamics simulations on the kinase domain were applied to elucidate differences in breathing dynamics between wild type and mutants of the DYGψ motif. Results: Our cellular approaches revealed that the kinase inhibitors MLi-2 and rebastinib both freeze the kinase domain in a stable conformation, however, only MLi-2 resembles an active conformation and induces filament formation. LRRK2RCKW showed, regardless of the mutation it was combined with, filament formation, indicating a shielding function of the N-terminal domains. This shielding function is impaired for pathogenic mutations in full length LRRK2. LRRK2RCKW retained kinase activity similar to full-length LRRK2. HDX-MS provided a comprehensive allosteric portrait of the kinase domain and revealed how MLi-2 binding is sensed by the entire protein. Molecular Dynamics simulations suggest that, while all domains contribute to regulating kinase activity and spatial distribution, it is the highly dynamic kinase domain, driven by the DYGψ motif, that coordinates the overall domain crosstalk and serves as a regulatory hub for the intrinsic regulation of LRRK2.Conclusion: These studies confirm our hypothesis that the N-terminal scaffolding domains shield the catalytic domains in an inactive state. PD mutations, MLi-2, or Rab GTPases can all unleash the catalytic domains while the active kinase conformation, but not kinase activity, is essential for docking onto microtubules.

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

“…PD mutations that lead to activation obviously alter the equilibrium between the active and inactive states. We showed previously how the two PD mutations in the kinase domain (G2019S and I2020T) unleash the inhibitory NTDs, and with our HDX-MS analysis of the kinase domain we showed how the disordered region surrounding the AS becomes more ordered by the binding of MLi-2 [12].…”

“…The two clusters of PD mutations highlight the importance of the COR-B:ROC domain interface and the hinging motion of the kinase domain while all of the mutations potentially "unleash" the inhibition that is imposed by the NTDs. The dominant organizing motif in COR-B is the Dk-Helix while the dominant motif in the kinase domain is the DYGψ motif as described previously [12]. With HDX-MS and GaMD we are beginning to achieve a deeper molecular understanding of these critical domain:domain interfaces as well as loop dynamics, which all contribute to the allosteric regulation of LRRK2 and are perturbed by mutations that make LRRK2 a risk factor for PD.…”

“…Hydrogen-deuterium exchange mass spectrometry LRRK2 RCKW proteins were expressed and purified from Sf9 cell [12]. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) was performed using a Waters Synapt G2Si equipped with nanoACQUITY UPLC system with H/DX technology and a LEAP autosampler.…”

“…Our overall HDX-MS coverage of LRRK2 RCKW , which was >98% (S1 Fig) , shows the relative fractional deuterium uptake of each peptide. We previously mapped the HDX-MS profile onto a model of the kinase domain [12] while here we mapped the HDX-MS exchange pattern onto the entire LRRK2 RCKW cryo-EM structure. This allows us to capture the crosstalk between and within the C-terminal domains.…”

Impact of Type II LRRK2 inhibitors on signalling and mitophagy

“…Three Type II kinase inhibitors have been reported to target LRRK2 namely GZD-824 [41], Rebastinib [45], and Ponatinib [46], compounds originally developed as inhibitors of the breakpoint cluster region-Abelson (Bcr-Abl) kinase to overcome clinically acquired mutationinduced resistance against imatinib the first line treatment of chronic myelogenous leukemia [43,[47][48][49]. These compounds also suppressed binding of LRRK2 to microtube filaments, consistent with Type II inhibitors maintaining LRRK2 in an inactive open conformation incapable of interacting with microtubules [41,45].…”