Structure of the ROC domain from the Parkinson's disease-associated leucine-rich repeat kinase 2 reveals a dimeric GTPase

Abstract

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of Parkinson's disease (PD). LRRK2 contains a Ras of complex proteins (ROC) domain that may act as a GTPase to regulate its protein kinase activity. The structure of ROC and the mechanism(s) by which it regulates kinase activity are not known. Here, we report the crystal structure of the LRRK2 ROC domain in complex with GDP-Mg(2+) at 2.0-A resolution. The structure displays a dimeric fold generated by extensive domain-swapping, resulting in a pair of active sites constructed with essential functional groups contributed from both monomers. Two PD-associated pathogenic residues, R1441 and I1371, are located at the interface of two monomers and provide exquisite interactions to stabilize the ROC dimer. The structure demonstrates that loss of stabilizing forces in the ROC dimer is likely related to decreased GTPase activity resulting from mutations at these sites. Our data suggest that the ROC domain may regulate LRRK2 kinase activity as a dimer, possibly via the C-terminal of ROC (COR) domain as a molecular hinge. The structure of the LRRK2 ROC domain also represents a signature from a previously undescribed class of GTPases from complex proteins and results may provide a unique molecular target for therapeutics in PD.

Fig. 1.

The unique dimeric ROC GTPase. (A) Stereoview of the domain-swapped dimer. The two individual monomers are shown in yellow and green. The GDP-Mg²⁺ ligands are shown in ball-and-stick format. (B) Ribbon representation of a single monomer. The three head, neck, and body subdomains are indicated, along with the labeled secondary structures. The P-loop, G3/Switch II, and G4 and G5 loops are indicated in orange, pink, red, and cyan, respectively. The disordered G2 loop is shown as a black dotted curve. (C) Surface representation highlighting the GDP-Mg²⁺ binding pocket on the surface of the dimer that is contributed from both monomers. The pair of functional units are shown as ROCs1 and ROCs2, respectively.

Fig. 2.

Structural basis of PD-associated mutations in ROC. (A) R1441 and W1434 from one monomer together with F1401 and P1406 from the other stack on each other alternately, forming a hydrophobic zipper at the dimer interface. The guanidinium group of R1441 also is hydrogen-bonded with the backbone carbonyl oxygen of F1401 and the hydroxyl group of T1404 on helix α2 from the other peptide chain. 2mF_o − DF_c electron density map is shown in blue. (B) I1371 is inserted in a hydrophobic cavity, which is constructed by residues from both monomers at the dimer interface. I1371 is shown in stick format and colored in orange. The surrounding residues are shown in stick format within the semitransparent surface representation. The color scheme is the same as that in Fig. 1. Note the side-chain methyl group of T1404 is pointing directly to the tip of I1371, forming a favorable van der Waals' interaction. (C) R1441C (lane 3), as a prototypical mutation at the dimer interface, decreases interaction with the full-length wild-type LRRK2 protein compared with wild-type GST fusions (lane 2); no interaction was seen with GST alone (lane 1). (D) Pull-down assays were quantified and corrected for the amount of LRRK2 protein in the inputs (middle blots). *, P < 0.0001; **, P < 0.01 compared with GST alone (one-way ANOVA with Student–Newman–Kuell's post hoc test; n = 3).

Fig. 3.

The nucleotide-binding site and a stereoview of the GDP-Mg²⁺ binding site. The GDP-Mg²⁺ ligand is shown in ball-and-stick format. The Mg²⁺ is colored in purple, and its direct water ligands are colored in cyan. The essential interacting residues from the dimer ROC are shown in stick format and are colored with the same scheme as in Fig. 1.

Fig. 4.

LRRK2 kinase regulation by ROC. (A) Stereo model of the LRRK2 functional GTPase unit (yellow and green for each monomer) superimposed on Ras (PDB ID 1ctq, red). Two key residues important in the regulation of Ras activity after GTP binding are Q61 and G12 (shown in red, labeled in red); the equivalent residues in LRRK2 are R1398 and T1343, respectively (shown in yellow, labeled in black). (B) Ras-like mutations R1398Q and T1343G were made individually and together (TQ/RG, lane 5) in the full-length LRRK2 construct, and their effects on autophosphorylation of the kinase were tested. (Upper) Autoradiogram with ³²P. (Lower) A blot for LRRK2. Quantification (n = 3, graph) shows that although each Ras-like mutation alone has no effect on kinase activity, the double TG/RQ caused a decrease in net kinase activity. **, P < 0.01; ***, P < 0.001 by ANOVA with Bonferroni's multiple comparison test. (C) ROC domain has intrinsic association with COR domain. Truncated domain proteins used for interaction assay are as follows: ROC–COR–kinase protein (lane 1), COR domain only (lane 2), and kinase domain only (lane 3), respectively, blotted with a myc tag. Their corresponding pull-down results with ROC domain as a GST fusion protein are shown in lanes 4–6, respectively. GST was used as negative control in lanes 7–9. (D) Model for ROC regulation of kinase activity. Dimeric ROC GTPase (yellow and green ovals) act as binary switches in regulation of kinase activation. Upon binding of GTP (stars), the activated ROC induces dimerization of the COR domain (barrels). The dimerization of COR domain further induces self-association of kinase domain, resulting in its autophosphorylation and activation (red ovals). Through hydrolysis of GTP to GDP (black oval), conformational changes in ROC disrupt the dimeric association of COR and kinase domain, inactivating the kinase (blue ovals). GTPase activating protein (GAP) and guanine nucleotide exchange factor (GEF) are not known.