UC San Diego

Mutations in the Leucine Rich Repeat Kinase 2 (LRRK2) are the most common cause of familial and sporadic Parkinson’s Disease. LRRK2 is a 286kDa multidomain protein, containing two catalytic domains: a Ras-like GTPase (ROC) and a Kinase domain. LRRK2 has been reported to localize to the cytosol in its inactive form and co-localize with membranes, where it becomes activated and carries out its function of phosphorylating its Rab substrates. For LRRK2 to perform this function, it must undergo conformational changes where LRRK2 is converted from its inactive form, in which the LRR is docked on top of the kinase domain blocking its access to the activation site, to an undocked LRR configuration where the active site is available for a Rab substrate to bind and undergo catalysis. This regulation of LRRK2 has been suggested to be the result from the intercommunication of the two catalytic domains, in which the ROC domain must exchange GDP for GTP to undock the LRR from the kinase domain, allowing the kinase domain to be accessible and activate subsequently. This thesis reports high-resolution CryoEM structures, in combination with cell-based and in vitro assays, to explore the roles of the Kinase and ROC domain in the activation of LRRK2. Chapter 2 focuses on the structural determination of full-length LRRK2 and the C-terminal half, known as LRRK2RCKW (ROC,COR, kinase and WD40 domains), using type I and II kinase inhibitors. The results reveal differences in active site of the most common familial mutation G2019S compared to wild-type LRRK2, structurally demonstrating its resistant and providing the basis for drug targeting of the mutant. We also show that LRRK2 N-terminal domain must fully undock from the kinase domain for it to close and adopt the active conformation, as even with the presence of a type I kinase inhibitor, full-length LRRK2 cannot fully close its kinase domain compared to LRRK2RCKW. Chapter 3 investigates the role of the ROC domain in modulating LRRK2’s activation via ROC nucleotide exchange, resulting in the first high resolution structure of active LRRK2 bound to a GTP analog, alongside multiple high-resolution structures of active and autoinhibited states. We also characterized LRRK2 bound to different Designed Ankyrin Repeat Proteins (DARPins) and obtained cellular and biochemical assay data showing that different DARPins can shift the equilibrium of LRRK2 from its closed, autoinhibited state to its open, active state. Our results revealed how LRRK2 activation is dependent on the nucleotide exchange of its ROC domain, providing the first active structure of full-length LRRK2. Cell and biochemical assays showed that LRRK2 mutants hyperactivation occurs through different parts of the activation pathway, where ROC-COR mutants lead to undocking of the N-terminus domain, and the kinase mutant G2019S acts directly on the kinase domain to increase its activity. Together, the work presented in this thesis provides new insights into the mechanism of LRRK2 activation and provides a framework for therapeutic development.