Structural and biochemical basis of ROC-dependent activation of LRRK2

Abstract Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson’s disease, yet the molecular mechanism governing LRRK2 activation remains incompletely understood. LRRK2 is a large multidomain enzyme whose kinase activity is regulated by intramolecular interactions and by its Ras of complex proteins (ROC) GTPase domain. Here, we combine cryo–electron microscopy, X-ray crystallography, and structure-guided biochemical perturbations to define how ROC conformational switching regulates LRRK2 activation. Cryo-EM reconstructions reveal that monomeric full-length LRRK2 samples three distinct conformational states—autoinhibited, intermediate, and activated—indicating that large-scale activation-associated rearrangements can occur through an intrinsic intramolecular pathway, independently of Rab29 binding, higher-order oligomerization, or membrane association. A 1.6 Å crystal structure of an extended ROC construct reveals intrinsic conformational plasticity within the GTPase switch regions that likely underlies these transitions. Structure-guided disulfide engineering identifies a functional coupling between residue R1441 and Switch II that directly modulates GTPase activity in both isolated ROC and full-length LRRK2. Disruption of this coupling phenocopies the disease-associated R1441H mutation. Together, these findings establish ROC as a dynamic conformational engine that drives a multistep intramolecular activation mechanism in LRRK2, providing mechanistic insight into how pathogenic mutations promote aberrant kinase activation.