Mutation-Driven Remodeling of the LRRK2 Kinase Free-Energy Landscape and Its Consequences for Conformational...

Parkinson's disease (PD), the second most common age-related neurodegenerative disorder worldwide, is associated with mutations in several genes, one of which is LRRK2 (leucine-rich repeat kinase 2). Despite cryo-EM structures elucidating the active and inactive architectures of the wild type (WT), the regulatory mechanisms and intermediate conformational states (INTs) that facilitate the transition between these forms, as well as the distinctions in the G2019S mutant, remain unclear. Here, we elucidated how mutations reshape the conformational dynamics through molecular dynamics simulations, highlighting the salt bridge contributions of the E1920-R2026 and K1906-D2017 residue pairs and fluctuations of the activation loop (A-loop) lid in stabilizing specific states. Furthermore, the reconstructed two-dimensional Well-Tempered Metadynamics (2D WT-MetaD) free-energy landscape along A-loop and K1906-E1920 coordinates revealed two distinct activation/inactivation pathways. Interestingly, the inactivation pathway in WT revealed a metastable "semi-open" state, while the G2019S mutation favored activation by decreasing the inactive-to-active energy barrier; however, once the active state was formed, it became comparatively stable and required more energy for the active-to-inactive transition than the WT. Thus, the G2019S mutation facilitated activation and remained in its active form, providing a mechanistic basis for its uncontrolled kinase hyperactivity. To reinforce the MetaD-derived stability of the G2019S active conformation over WT, we assessed the binding of the type-I inhibitor LRRK2-IN-1 for active state conformational rearrangements in the ligand-bound state. The absolute binding free-energy consistently showed that the ligand preferentially stabilizes the mutant active state, highlighting its binding affinity and modestly increased inhibitor potency, relative to WT. Finally, two-dimensional Umbrella Sampling (2D-US) showed apo-state transitions arising from DYGI (D2017-Y2018-G2019-I2020) rearrangements and αC-helix coupling via COM distances of K1906-E1920 and E1920-R2026 residue pairs. The results revealed that the mutant (G2019S) follows two routes with four INTs.