Capturing the domain crosstalk in full length LRRK2 and LRRK2(RCKW)

LRR-kinase 2 (LRRK2) is a multi-domain protein with three catalytically inert N-terminal domains (NtDs) and four C-terminal domains, including a kinase and a GTPase domain. LRRK2 mutations are linked to Parkinson's Disease (PD). Recent structures of LRRK2(RCKW) and a full-length inactive LRRK2 (fl-LRRK2(INACT)) monomer revealed that the kinase domain drives LRRK2 activation. The LRR domain and also an ordered LRR–COR linker, wrap around the C-lobe of the kinase domain and sterically block the substrate binding surface in fl-LRRK2(INACT). Here, we focus on the crosstalk between domains. Our biochemical studies of GTPase and kinase activities of fl-LRRK2 and LRRK2(RCKW) reveal how mutations influence this crosstalk differently depending on the domain borders investigated. Furthermore, we demonstrate that removing the NtDs leads to altered intramolecular regulation. To further investigate the crosstalk, we used Hydrogen–Deuterium exchange Mass Spectrometry (HDX-MS) to characterize the conformation of LRRK2(RCKW) and Gaussian Accelerated Molecular Dynamics (GaMD) to create dynamic portraits of fl-LRRK2 and LRRK2(RCKW). These models allowed us to investigate the dynamic changes in wild-type and mutant LRRK2s. Our data show that the α3(ROC) helix, the Switch II motif in the ROC domain, and the LRR–ROC linker play crucial roles in mediating local and global conformational changes. We demonstrate how these regions are affected by other domains in fl-LRRK2 and LRRK2(RCKW) and show how unleashing of the NtDs as well as PD mutations lead to changes in conformation and dynamics of the ROC and kinase domains which ultimately impact kinase and GTPase activities. These allosteric sites are potential therapeutic targets.