Abstract

Leucine-rich repeat kinase 2 (LRRK2) is a large multidomain protein, and LRRK2 mutants are recognized risk factors for Parkinson's disease (PD). Although the precise mechanisms that control LRRK2 regulation and function are unclear, the importance of the kinase domain is strongly implicated, since 2 of the 5 most common familial LRRK2 mutations (G2019S and I2020T) are localized to the conserved DFGψ motif in the kinase core, and kinase inhibitors are under development. Combining the concept of regulatory (R) and catalytic (C) spines with kinetic and cell-based assays, we discovered a major regulatory mechanism embedded within the kinase domain and show that the DFG motif serves as a conformational switch that drives LRRK2 activation. LRRK2 is quite unusual in that the highly conserved Phe in the DFGψ motif, which is 1 of the 4 R-spine residues, is replaced with tyrosine (DY2018GI). A Y2018F mutation creates a hyperactive phenotype similar to the familial mutation G2019S. The hydroxyl moiety of Y2018 thus serves as a "brake" that stabilizes an inactive conformation; simply removing it destroys a key hydrogen-bonding node. Y2018F, like the pathogenic mutant I2020T, spontaneously forms LRRK2-decorated microtubules in cells, while the wild type and G2019S require kinase inhibitors to form filaments. We also explored 3 different mechanisms that create kinase-dead pseudokinases, including D2017A, which further emphasizes the highly synergistic role of key hydrophobic and hydrophilic/charged residues in the assembly of active LRRK2. We thus hypothesize that LRRK2 harbors a classical protein kinase switch mechanism that drives the dynamic activation of full-length LRRK2.

Highlights

  • Combining the concept of regulatory (R) and catalytic (C) spines with kinetic and cell-based assays, we discovered a major regulatory mechanism embedded within the kinase domain and show that the DFG motif serves as a conformational switch that drives Leucine-rich repeat kinase 2 (LRRK2) activation

  • In LRRK2, the kinase domain is embedded in a large multidomain protein, and much is still unknown about the biological role of LRRK2 and how it is activated

  • Using a combination of structure-based analysis and site-directed mutagenesis accompanied with cell-based microtubule decoration assays, we discovered a previously unappreciated regulatory mechanism for the LRRK2 kinase domain

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Summary

Introduction

3 of the 5 most common pathological, familial mutants of LRRK2 (R1441C, Y1699C, I2020T) are predicted to form filamentous structures that represent LRRK2 docked onto microtubules [12]. Until now, it is not clear if the docking onto microtubules is associated with the pathogenicity of those mutations. The hydroxyl moiety of Y2018 serves as a “brake,” stabilizing the inactive conformation; removing it destroys a key inhibitory hydrogen-bonding node These data reveal an LRRK2-specific regulatory mechanism, confirming that the kinase domain functions as a classical kinase that controls overall conformational dynamics in fulllength LRRK2 and drives therapeutic strategies

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