Aoto, P. C. | bioRxiv Science

bioRxiv2021

Two helices control the dynamic crosstalk between the catalytic domains of LRRK2

The two major molecular switches in biology, kinases and GTPases, are both contained in the Parkinsons Disease-related Leucine-rich repeat kinase 2 (LRRK2). Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and Molecular Dynamics (MD) simulations, we generated a comprehensive dynamic allosteric portrait of the C-terminal domains of LRRK2 (LRRK2RCKW). We identified two helices that shield the kinase domain and regulate LRRK2 conformation and function. One docking helix in COR-B (Dk-Helix) tethers the COR-B domain to the C helix of the kinase domain and faces its Activation Loop, while the C-terminal helix (Ct-Helix) extends from the WD40 domain and interacts with both kinase lobes. The Ct-Helix and the N-terminus of the Dk-Helix create a "cap" that regulates the N-Lobe of the kinase domain. Our analyses reveal allosteric sites for pharmacological intervention and confirm the kinase domain as the central hub for conformational control.

biochemistry↗

bioRxiv2020

Conformation and dynamics of the kinase domain drive subcellular location and activation of LRRK2

In a multi-tiered approach, we explored how Parkinsons Disease-related mutations hijack the finely tuned activation process of Leucine-Rich Repeat Kinase 2 (LRRK2) using a construct containing the ROC, Cor, Kinase and WD40 domains (LRRK2RCKW). We hypothesized that the N-terminal domains shield the catalytic domains in an inactive state. PD mutations, type-I LRRK2 inhibitors, or physiological Rab GTPases can unleash the catalytic domains while the active kinase conformation, but not kinase activity, is essential for docking onto microtubules. Mapping solvent accessible regions of LRRK2RCKW employing hydrogen-deuterium exchange mass spectrometry (HDX-MS) revealed how inhibitor binding is sensed by the entire protein. Molecular Dynamics simulations of the kinase domain elucidated differences in conformational dynamics between wt and mutants of the DYG{psi} motif. While all domains contribute to regulating kinase activity and spatial distribution, the kinase domain, driven by the DYG{psi} motif, coordinates domain crosstalk and serves as an intrinsic hub for LRRK2 regulation.

biochemistry↗