Markov State Models and Molecular Dynamics Simulations Provide Understanding of the Nucleotide-Dependent Dimerization-Based Activation of LRRK2 ROC Domain

Abstract

Mutations in leucine-rich repeat kinase 2 (LRRK2) are recognized as the most frequent cause of Parkinson's disease (PD). As a multidomain ROCO protein, LRRK2 is characterized by the presence of both a Ras-of-complex (ROC) GTPase domain and a kinase domain connected through the C-terminal of an ROC domain (COR). The bienzymatic ROC-COR-kinase catalytic triad indicated the potential role of GTPase domain in regulating kinase activity. However, as a functional GTPase, the detailed intrinsic regulation of the ROC activation cycle remains poorly understood. Here, combining extensive molecular dynamics simulations and Markov state models, we disclosed the dynamic structural rearrangement of ROC's homodimer during nucleotide turnover. Our study revealed the coupling between dimerization extent and nucleotide-binding state, indicating a nucleotide-dependent dimerization-based activation scheme adopted by ROC GTPase. Furthermore, inspired by the well-known R1441C/G/H PD-relevant mutations within the ROC domain, we illuminated the potential allosteric molecular mechanism for its pathogenetic effects through enabling faster interconversion between inactive and active states, thus trapping ROC in a prolonged activated state, while the implicated allostery could provide further guidance for identification of regulatory allosteric pockets on the ROC complex. Our investigations illuminated the thermodynamics and kinetics of ROC homodimer during nucleotide-dependent activation for the first time and provided guidance for further exploiting ROC as therapeutic targets for controlling LRRK2 functionality in PD treatment.

Keywords: Markov state models; Parkinson’s disease; Ras-of-complex GTPase domain; leucine-rich repeat kinase 2 (LRRK2); molecular dynamics (MD) simulations; network analysis.

Figure 1

(A) Domain architecture of human LRRK2 with its respective amino acid positions. ARM: Armadillo repeats region; ANK: Ankyrin repeat region; LRR: leucine-rich repeats; ROC: Ras-of-complex GTPase domain; COR: C-terminal-of-ROC domain; Kinase: protein tyrosine kinase-like domain; WD40: WD40 repeat. The detailed functional partition of ROC is depicted above with the guanine nucleotide phosphate-binding loop (P-loop), Switch I and Switch II, and G4 and G5 loop highlighted in green rectangles. The residue position R1441 is highlighted with a red arrow. (B) Cartoon model of ROC monomer roughly divided into head (including β1, α1, β2, and β3), neck (including α2) and body (including β4, α3, β5, α4, β6, and α5) subdomains. The monomer is colored based on a secondary structure, and the P-loop, Switch I, Switch II, G4 loop, and G5 loop are highlighted in light green, hot pink, purple, brown, and gold, respectively. Residue R1441 is highlighted. (C) Schematic model of ROC dimer–monomer dynamic transition during nucleotide turnover based on GAD theory. (D) Cartoon model of ROCs-GDP, the two monomers are shown in pink (ROC^A) and green (ROC^B), respectively. (E) Molecular surface representation of ROC dimer highlighting the nucleotide-binding pockets (blue-dashed oval). Gnt1 is accommodated by ROC^A head and ROC^B body, while Gnt2 interacts with ROC^A body and ROC^B head. Unless otherwise specified, the graphs showing ROCs overview in our work will all be presented from this perspective with Gnt1 residing on the top-left. The presumed compact functional units are denoted as ROCs1 and ROCs2.

Figure 2

Representative structures of the four metastable states in ROCs-GDP~ROCs-GTP MSMs: (A) S1_GDP for macrostate S1, (B) S2_GTP for macrostate S2, (C) S3_GDP for macrostate S3, and (D) S4_GTP for macrostate S4. Structural segments that exhibited significant differences are highlighted in the opaque cartoon, with the remaining being transparent. (E) Distribution of the four metastable states in Rocs-GDP~ROCs-GTP MSMs with the portion of each macrostate. (F) Transition time between the macrostates calculated by mean first passage time (MFPT). OTHER: denoted the transition between S2_GTP/S3_GDP and all other remaining metastable states.

Figure 3

Detailed bonding network between the guanine nucleotide and ROCs in the representative structure from (A) ROCs-GDP, S3_GDP, and (B) ROCs-GTP, S2_GTP. The molecular surface diagram in the middle highlighted regions implicated in nucleotide binding, as color-coded in Figure 1B. The zoom-in graphs above (i) and below (ii) depicted the detailed interactions between ROCs and nucleotide Gnt1 and Gnt2, respectively.

Figure 4

Generalized cross correlation matrix (GCCM) of (A) ROCs-GDP and (B) ROCs-GTP. The colormap used for the graph is shown on the right with no/weak correlations indicated in green and strong ones in magenta. The correlation between ROC^A head and ROC^B body, and that between ROC^A body and ROC^B head subdomain, is highlighted in red and blue dashed line rectangles, respectively. Cartoon representation of (C) ROCs-GDP and (E) ROCs-GTP color-coded based on their corresponding community structures. Simplified community network map with inter-community crosstalk in (D) ROCs-GDP and (F) ROCs-GTP. Each community in the original structure is reduced to a sphere whose area is proportional to the residue components. Inter-community crosstalk is visualized by sticks of width that are proportional to edge connectivity between communities.

Figure 5

Distribution of metastable states in (A) ROCs^R1441C-GDP~ROCs-GTP (5 macrostates), (B) ROCs^R1441G-GDP~ROCs-GTP (5 macrostates), and (C) ROCs^R1441H-GDP~ROCs-GTP (4 macrostates) MSM. The type of nucleotide ligand for ROCs is indicated with subscripts, and the percentage of each macrostate is shown beside the corresponding colormap. MFPT between the predominant GDP-bound ROCs mutant and GTP-bound ROCs calculated in (D) ROCs^R1441C-GDP~ROCs-GTP, (E) ROCs^R1441G-GDP~ROCs-GTP, and (F) ROCs^R1441H-GDP~ROCs-GTP MSM. The two-ended black arrow between metastables and OTHER denoted the transition between the implicated macrostates and all other remaining metastable states in the corresponding MSM.

Figure 6

Simplified community network map with inter-community crosstalk in (A) ROCs^R1441C-GDP, (C) ROCs^R144G-GDP, and (E) ROCs^R144H-GDP. Cartoon diagrams with residues color-coded by their corresponding community partition were made transparent as the background to indicate the spatial position of each community. A schematic diagram of allosteric signaling between the mutated site at position 1441 and the nucleotide-binding motifs Gnt1 and Gnt2 (marked with cyan and orchid dotted lines) in (B) ROCs^R1441C-GDP, (D) ROCs^R144G-GDP, and (F) ROCs^R144H-GDP mutants. Secondary structures of ROC^A are represented by colored hollow rectangles, and ROC^B components are shown in filled ovals. The gray and pink arrows, respectively, indicate allosteric signals from the mutation site on ROC^A and ROC^B, and black arrows denote paths involved in transmitting signals from both mutation sites. Note that all arrows here are one-ended for clarity; the actual information flow can pass in both directions for reversible communication.