The In situ Structure of Parkinson's Disease-Linked LRRK2

Abstract

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of familial Parkinson's disease (PD). LRRK2 is a large multi-domain protein containing a kinase and GTPase surrounded by multiple domains involved in protein interactions. LRRK2 involved in various biological processes including cytoskeletal integrity, membrane trafficking, autophagy, and mitochondrial homeostasis. LRRK phosphorylates a subset of Rab GTPases and modulates their membrane localization and function. Most of the PD pathogenic mutations are found in the catalytic domains, but all result in hyperactivation of the kinase. This suggests that crosstalk between the GTPase and kinase domains is critical for the regulation of LRRK2 kinase activity. Hyperactivation of LRRK2's kinase is also reported in non-familial PD cases, suggesting that suppression of kinase activity might be beneficial for a wide range of PD patients. Despite being a promising drug target, LRRK2 has eluded structural determination due to the difficulties to purify active full-length LRRK2 protein from cells. Interestingly, LRRK2 has been shown to form filamentous structures surrounding microtubules in cells and several PD mutants or treatment of LRRK2 kinase inhibitor enhance its filament formation. Using in situ cryo-electron tomography and subtomogram averaging, we reveal a 14-Å structure of LRRK2 bearing a pathogenic mutation that oligomerizes as a right-handed double-helix around microtubules, which are left-handed. Using integrative modeling, we determine the architecture of LRRK2, revealing that the GTPase points towards the microtubule, while the kinase is exposed to the cytoplasm. We identify several oligomerization interfaces mediated by non-catalytic regions of LRRK2. Mutation of one of these abolishes LRRK2 microtubule-association in cells. Our work demonstrates the power of cryo-electron tomography to obtain structures of previously unsolved proteins in their cellular environment and provides insights into LRRK2 function and PD pathogenicity.