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-Å 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. Parkinson's disease (PD) is a common, age-related neurodegenerative disorder for which only symptomatic treatment is available. The etiology of PD is poorly understood, but over the past decade it has become clear that there are rare families with Mendelian inheritance (1). Of the genes that cause PD, dominantly inherited mutations in leucine-rich repeat kinase 2 (LRRK2) are numerically the most common and account for an appreciable fraction of apparently sporadic PD (reviewed in ref.2). LRRK2 encodes a large (2,527-aa) multidomain protein originally identified as a unique kinase with leucine-rich repeats. LRRK2 is a member of a superfamily of proteins, named ROCO, that includes at least three other human proteins: leucine-rich repeat kinase 1 (LRRK1), death-associated protein kinase (DAPK1), and malignant fibrous histiocytoma-amplified sequences with leucine-rich tandem repeat-1 (MASL1) (3). A unique feature of all ROCO proteins is a 200-to 250-aa Ras-related GTPase or Ras of complex proteins (ROC) domain, followed by a C-terminal of ROC (COR) domain immediately before the kinase domain.Both LRRK2 and LRRK1 have been shown to be active protein kinases in vitro (4-7), and some mutations are found in the kinase domain. These mutations generally increase kinase activity, although there are some discrepancies in different studies as to whether all mutations increase kinase function (4,6,(8)(9)(10)(11). However, the kinase activity of LRRK2 is required for the ability of the mutant protein to cause neuronal damage, at least in cell culture models (5, 10), suggesting that kinase inhibitors may represent a therapeutic avenue for PD.Although the kinase domain therefore is important in understanding pathogenesis, mutations also are fo...

Update on the Functional Biology of Lrrk2

“…GTPase activity has been more difficult to demonstrate [57,60], presumably owing to the requirement of GEFs and GAPs for in vitro assays; however, optimized experimental conditions have now enabled GTPase activity, albeit low, to be detected [33,53,58,59]. Several groups now agree that Lrrk2 Roc domain mutations do not increase GTP binding [33,58,59], but Roc mutants do reduce GTPase activity [33,58,59] and suggest that Roc domain mutations prolong Lrrk2 in the active GTP-bound state.…”

“…Whilst support for kinase domain dimeric interactions are lacking [44,51], the Roc domain is able to interact with itself and with the central Roc-COR-kinase region of full-length Lrrk2, an interaction that is strengthened by the WD40 domain [51,53]. Roc-Roc interaction is not nucleotide dependent, but it is weakened by the R1441C pathogenic mutation, indicating that destabilizing dimeric interactions may be important in aberrant Lrrk2 GTPase activity [53]. The caveat is that the I1371V mutation, used to support this hypothesis, is not pathogenic but a benign polymorphism found at appreciable frequency in elderly controls [Owen Ross, Pers.…”

“…Several groups now have shown that Lrrk2 can bind GTP via GTP-sepharose or GTPγS-binding assays, whereas disruption of the P-loop in the nucleotide-binding pocket of the Roc domain abolishes GTP binding [33,47,53,[57][58][59][60]. GTPase activity has been more difficult to demonstrate [57,60], presumably owing to the requirement of GEFs and GAPs for in vitro assays; however, optimized experimental conditions have now enabled GTPase activity, albeit low, to be detected [33,53,58,59].…”

“…In human lymphoblastoid cell lines, endogenous human Lrrk2 appears to dimerize, with the presence of approximately 600 kDa peak and higher molecular weight complex in immunoblots [51]. Several in vitro studies studying exogenous Lrrk2 have reported the ability to dimerize and involve multiple domain swapping [44,[51][52][53]. Whilst support for kinase domain dimeric interactions are lacking [44,51], the Roc domain is able to interact with itself and with the central Roc-COR-kinase region of full-length Lrrk2, an interaction that is strengthened by the WD40 domain [51,53].…”

“…Several in vitro studies studying exogenous Lrrk2 have reported the ability to dimerize and involve multiple domain swapping [44,[51][52][53]. Whilst support for kinase domain dimeric interactions are lacking [44,51], the Roc domain is able to interact with itself and with the central Roc-COR-kinase region of full-length Lrrk2, an interaction that is strengthened by the WD40 domain [51,53]. Roc-Roc interaction is not nucleotide dependent, but it is weakened by the R1441C pathogenic mutation, indicating that destabilizing dimeric interactions may be important in aberrant Lrrk2 GTPase activity [53].…”