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

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 �nely 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 (LRRK2 RCKW ). We focused on the biochemical characterization of LRRK2 RCKW 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 LRRK2 RCKW 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 �lament formation. LRRK2 RCKW showed, regardless of the mutation it was combined with, �lament formation, indicating a shielding function of the N-terminal domains. This shielding function is impaired for pathogenic mutations in full length LRRK2. LRRK2 RCKW 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 When testing Rab8a as a substrate for the LRRK2 RCKW construct employing Western blotting against pT72 and the His-tag of His-Rab8a, we revealed increased phosphorylation of Rab8a by LRRK2 RCKW Y2018F, G2019S and Y1699C. MLi-2 was shown to e�ciently block phosphorylation of Rab8a which was also found for the kinase dead mutant D2017A. Quanti�cation was performed for two independent Western Blots using two independent protein preparations. For each quanti�cation, the pT72 signals were referenced to the signal for the His-tag of 6xHis-Rab8a and then normalized to the resulting wt signal. The dotted line therefore represents 100% of the wt signal. approach focusing on the importance of the kinase domain. Our �rst layer concentrated on the spatial and temporal distribution of full-length LRRK2 in a cellular context as a function of the high a�nity kinase inhibitor, MLi-2, which provided us with a real-time assay for �lament formation in live cells. The effects of removing the N-terminus on cellular distribution was then explored with our LRRK2 RCKW variants. These studies led to the prediction that the N-terminal scaffolding domains shield the catalytic domains in the inactive resting state of LRRK2. The 2nd layer focused on the biochemical characterization of LRRK2 RCKW variants by demonstrating substrate-speci�c kinase activity. Here we showed that the LRRK2 RCKW protein retained the catalytic machinery for mediating phosphoryl transfer. In the next layer we used HDX-MS analysis of LRRK2 RCKW to provide a portrait of the conformational states of LRRK2 RCKW in the presence and absence of MLi2. Mapping the solvent accessible regions in a model of LRRK2 RCKW not only provides an allosteric portrait of the kinase domain but also suggests multidomain crosstalk. Finally, we performed GaMD calculations on the LRRK2 kinase domain to elucidate at a molecular level the differences in breathing dynamics between LRRK2 wt and the pathogenic kinase domain mutations Y2018F, G2019S and I2020T, explicitly establishing the role of the DYGψ motif as a dynamic regulator. With this multi-scale approach we were able to clearly demonstrate that the kinase activity and the spatial distribution of LRRK2 is not only regulated by single domains but by a complex interplay of all the embedded protein domains. The highly dynamic kinase domain, nevertheless, seems to play a crucial role in coordinating the overall domain crosstalk and serves as a central regulatory hub for the intrinsic regulation of LRRK2.