Abstract
To explore how pathogenic mutations of the multidomain leucine-rich repeat kinase 2 (LRRK2) hijack its finely tuned activation process and drive Parkinson's disease (PD), we used a multitiered approach. Most mutations mimic Rab-mediated activation by "unleashing" kinase activity, and many, like the kinase inhibitor MLi-2, trap LRRK2 onto microtubules. Here we mimic activation by simply deleting the inhibitory N-terminal domains and then characterize conformational changes induced by MLi-2 and PD mutations. After confirming that LRRK2RCKW retains full kinase activity, we used hydrogen-deuterium exchange mass spectrometry to capture breathing dynamics in the presence and absence of MLi-2. Solvent-accessible regions throughout the entire protein are reduced by MLi-2 binding. With molecular dynamics simulations, we created a dynamic portrait of LRRK2RCKW and demonstrate the consequences of kinase domain mutations. Although all domains contribute to regulating kinase activity, the kinase domain, driven by the DYGψ motif, is the allosteric hub that drives LRRK2 regulation.
Highlights
| leucine-rich repeat kinase 2 (LRRK2) hydrogen-deuterium exchange mass | | spectrometry (HDX-MS) Gaussian accelerated molecular dynamics | kinase regulation Parkinson’s disease
In particular, on the DYGI motif in the kinase domain which is a hotspot for Parkinson’s disease (PD) mutations and a critical part of the switch mechanism that leads to LRRK2 activation [10]
To characterize and dissect the functional properties of the catalytic domains of LRRK2, in particular the kinase domain, we used a multiscale approach that extends from testing real-time filament formation in live cells to assessing the consequences of MD simulations of PD mutations in the kinase domain
Summary
| leucine-rich repeat kinase 2 (LRRK2) hydrogen-deuterium exchange mass | | spectrometry (HDX-MS) Gaussian accelerated molecular dynamics | kinase regulation Parkinson’s disease. LRRK2 dimerization, and interactions with other proteins such as Rabs and 14-3-3 proteins, further increase the complexity of LRRK2 regulation [11,12,13,14,15,16] To unravel this complexity, we used a multitiered approach beginning with a cell-based assay for filament formation, a process that correlates with LRRK2 docking onto MTs [6]. To achieve a mechanistic understanding of LRRK2, a multidomain protein kinase, we must understand how the conformational landscape is changed by specific mutations that cause LRRK2 to become a driver of Parkinson’s disease (PD) To meet this challenge, we used a construct, LRRK2RCKW, that lacks the N-terminal inhibitory domains. Our multitiered analysis defines the kinase domain as a dynamic allosteric hub for LRRK2 activation
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