Parkinson's disease-associated mutations in the GTPase domain of LRRK2 impair its nucleotide-dependent conformational dynamics

Abstract

Mutation in leucine-rich repeat kinase 2 (LRRK2) is a common cause of familial Parkinson's disease (PD). Recently, we showed that a disease-associated mutation R1441H rendered the GTPase domain of LRRK2 catalytically less active and thereby trapping it in a more persistently "on" conformation. However, the mechanism involved and characteristics of this on conformation remained unknown. Here, we report that the Ras of complex protein (ROC) domain of LRRK2 exists in a dynamic dimer-monomer equilibrium that is oppositely driven by GDP and GTP binding. We also observed that the PD-associated mutations at residue 1441 impair this dynamic and shift the conformation of ROC to a GTP-bound-like monomeric conformation. Moreover, we show that residue Arg-1441 is critical for regulating the conformational dynamics of ROC. In summary, our results reveal that the PD-associated substitutions at Arg-1441 of LRRK2 alter monomer-dimer dynamics and thereby trap its GTPase domain in an activated state.

Keywords: GTPase; Parkinson disease; Ras of complex proteins (ROC); conformational change; conformational dynamics; disease mutation; enzyme activation; kinase; leucine-rich repeat kinase 2 (LRRK2); molecular dynamics.

Figure 1.

The ROC constructs of LRRK2 under investigation. a, a schematic depiction of LRRK2 showing the boundaries of ROC_ext with respect to the other domains, and the sites of PD-associated mutations studied. b, SDS-PAGE of purified ROC_ext carrying the PD-associated mutations R1441H (H), R1441G (G), and R1441C (C), and a synthetic mutant R1441K (K).

Figure 2.

Biochemical properties of PD-associated mutations in the ROC domain of LRRK2 compared with the WT. a, GTPase activity, phosphate release against time, of WT (black line) ROC_ext and various mutants R1441C (orange), R1441G (gray), R1441H (blue), and R1441K (green). b, fluorescence polarization-based GDP binding assay of WT ROC_ext (black) and the PD-associated mutants R1441C/R1441G/R1441H (orange, gray, and blue, respectively). Affinity is shown in parentheses. c, fluorescence polarization-based GTP-binding assay of WT ROC_ext (black) and the PD-associated mutants R1441C/R1441G/R1441H (orange, gray, and blue, respectively). Affinity is shown in parentheses. d, fluorescence-based thermal denaturation assay of ROC_ext (black), R1441C (orange), R1441G (gray), and R1441H (blue). Melting temperature of shown in parentheses.

Figure 3.

Conformations of PD-associated mutations in the ROC domain of LRRK2 compared with the WT. a, CD spectrometry of WT (black line) ROC_ext and various mutants R1441C (orange), R1441G (gray), R1441H (blue). b, SEC-MALS of WT ROC_ext dimer (solid black line), WT monomer (dotted black line) along with the PD-associated mutants R1441C/R1441G/R1441H (orange, gray, and blue, respectively).

Figure 4.

Oligomeric states and GTPase activity of R1441Y. a, size-exclusion chromatography of the WT dimer (solid black line), WT monomer (dotted black line), and the R1441Y mutant (green line). b, GTPase activity, phosphate release against time, of the R1441Y mutant (green) compared with that of the WT (black).

Figure 5.

Nucleotide-dependent dimer–monomer equilibrium of ROC. a, size-exclusion chromatography of the WT ROC_ext (solid black line) incubated with 2.5 mm GTP (orange dashed line) and 5.0 mm GTP (red dashed line). b, the same experiment as a, but with 2.5 mm GDP (light green dashed line) and 5.0 mm GDP (green dashed line). c, dimer–monomer interconversion experiment showing that the same protein sample converting from mostly dimers (solid black line) to nearly completely monomers upon binding excess GTP (dashed black line) and then reverts back to forming some dimers upon binding to GDP (green dashed line), but the same treatment with GTP did not produce any dimers (red dashed line). d, the same experiment as c for the ROC_ext carrying the PD-associated mutation R1441G, showing a complete loss of nucleotide-dependent dimer–monomer interconversion.

Figure 6.

Effects of PD-associated mutations on LRRK2. a, cell-based subcellular localization assay showing that the disease-associated mutations (R1441H/R1441C/R1441G) that perturbed ROC dimerization cause accumulation of full-length LRRK2 at the trans-Golgi network. The symbols ** and *** denote p values 0.001 to 0.01 and 0.0001 to 0.001, respectively. b, representative images of the HEK293FT cells collected in the trans-Golgi localization assay shown in a. The 50 μm scale shown in the bottom right panel is representative of all other panels in the figure.

Figure 7.

Model of ROC dimer–monomer dynamic equilibrium summarizing our observations that GTP binding to dimeric ROC led to its dissociation into monomers. GDP binding, either by GTP hydrolysis or nucleotide exchange to GDP shifts the equilibrium to the dimeric conformation. The PD-associated mutations perturbs its GTPase activity and results in a prolonged on state.