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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.

SUBMITTER: Deng J

PROVIDER: S-EPMC2234173 | biostudies-literature | 2008 Feb

REPOSITORIES: biostudies-literature

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

Deng Junpeng J Lewis Patrick A PA Greggio Elisa E Sluch Eli E Beilina Alexandra A Cookson Mark R MR

Proceedings of the National Academy of Sciences of the United States of America 20080129 5

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-sw ...[more]

PMID: 18230735

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Project description:Recent studies have identified mutations in the leucine-rich repeat kinase2 gene (LRRK2) in the most common familial forms and some sporadic forms of Parkinson's disease (PD). LRRK2 is a large and complex protein that possesses kinase and GTPase activities. Some LRRK2 mutants enhance kinase activity and possibly contribute to PD through a toxic gain-of-function mechanism. Given the role of LRRK2 in the pathogenesis of PD, understanding the kinetic mechanism of its two enzymatic properties is critical for the discovery of inhibitors of LRRK2 kinase that would be therapeutically useful in treating PD. In this report, by using LRRK2 protein purified from murine brain, first we characterize kinetic mechanisms for the LRRK2-catalyzed phosphorylation of two peptide substrates: PLK-derived peptide (PLK-peptide) and LRRKtide. We found that LRRK2 follows a rapid equilibrium random mechanism for the phosphorylation of PLK-peptide with either ATP or PLK-peptide being the first substrate binding to the enzyme, as evidenced by initial velocity and inhibition mechanism studies with nucleotide analogues AMP and AMP-PNP, product ADP, and an analogue of the peptide substrate. The binding of the first substrate has no effect on the binding affinity of the second substrate. Identical mechanistic conclusions were drawn when LRRKtide was the phosphoryl acceptor. Next, we characterize the GTPase activity of LRRK2 with a k(cat) of 0.2 +/- 0.02 s(-1) and a K(m) of 210 +/- 29 microM. A SKIE of 0.97 +/- 0.04 was measured on k(cat) for the GTPase activity of LRRK2 in a D(2)O molar fraction of 0.86 and suggested that the product dissociation step is rate-limiting, of the steps governed by k(cat) in the LRRK2-catalyzed GTP hydrolysis. Surprisingly, binding of GTP, GDP, or GMP has no effect on kinase activity, although GMP and GDP inhibit the GTPase activity. Finally, we have identified compound LDN-73794 through screen of LRRK2 kinase inhibitors. Our study revealed that this compound is a competitive inhibitor of the binding of ATP and inhibits the kinase activity without affecting the GTPase activity.

Dataset Information

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

Publications

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

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