Structural biology of the LRRK2 GTPase and kinase domains: implications for regulation

Abstract

Human leucine rich repeat kinase 2 (LRRK2) belongs to the Roco family of proteins, which are characterized by the presence of a Ras-like G-domain (Roc), a C-terminal of Roc domain (COR), and a kinase domain. Mutations in LRRK2 have been found to be thus far the most frequent cause of late-onset Parkinson’s disease (PD). Several of the pathogenic mutations in LRRK2 result in decreased GTPase activity and enhanced kinase activity, suggesting a possible PD-related gain of abnormal function. Important progress in the structural understanding of LRRK2 has come from our work with related Roco proteins from lower organisms. Atomic structures of Roco proteins from prokaryotes revealed that Roco proteins belong to the GAD class of molecular switches (G proteins activated by nucleotide dependent dimerization). As in LRRK2, PD-analogous mutations in Roco proteins from bacteria decrease the GTPase reaction. Studies with Roco proteins from the model organism Dictyostelium discoideum revealed that PD mutants have different effects and most importantly they explained the G2019S-related increased LRRK2 kinase activity. Furthermore, the structure of Dictyostelium Roco4 kinase in complex with the LRRK2 inhibitor H1152 showed that Roco4 and other Roco family proteins can be important for the optimization of the current, and identification of new, LRRK2 kinase inhibitors. In this review we highlight the recent progress in structural and biochemical characterization of Roco proteins and discuss its implication for the understanding of the complex regulatory mechanism of LRRK2.

Structural model of the dimeric Parkinson’s protein LRRK2 reveals a compact architecture involving distant interdomain contacts

“…Seven unique domains have been identified from primary sequence of LRRK2, including three N-terminal repeat domains (armadillo, ankyrin, and LRR), followed by the RocCOR G-domain/dimerization interface, a kinase domain, and a C-terminal WD40 repeat (9). Single-domain crystal structures of the LRR (reported here), RocCOR, and kinase domains from LRRK2 homologs (e.g., a Roco protein from Chlorobium tepidum, Roco4 from Dictyostelium discoideum) are available and can be used in integrative modeling studies (35,36).…”

“…The structure of the LRR domain of the C. tepidum Roco protein (PDB ID code 5IL7), which served as a template to model the LRRK2 LRR domain, also contains the Roc α0-helix (36) at its C terminus, allowing the alignment of the LRR and RocCOR models at this position. Although bacterial Roco proteins lack the kinase domain, they can be used as a model for the LRRRocCOR region (9). Two different alignments for the LRRK2 and C. tepidum Roco LRR domains were tested for threading calculations (SI Materials and Methods), leading to two alternative LRR model structures.…”

“…LRRK2 is a multidomain, 286-kDa protein exhibiting both GTPase and kinase activities (5-7) that belongs to the recently identified Roco protein family of G proteins. Members of this family have a Ras of complex proteins (Roc) G-domain and an adjacent conserved C-terminal of Roc (COR) dimerization domain in common (8,9). Besides the enzymatic core region, LRRK2 contains four predicted solenoid domains commonly involved in proteinprotein interactions (10).…”

A High-Throughput Screen to Identify LRRK2 Kinase Inhibitors for the Treatment of Parkinson’s Disease Using RapidFire Mass Spectrometry

“…21 Overall, the issues described above are likely to result in a number of false positives or nonspecific hits, which will artificially inflate the overall hit rate of any screen for LRRK2. This is a particular issue given that it is a challenge to isolate the required quantity of high-quality recombinant LRRK2 protein, 22 and therefore it is essential that reagent not be wasted in confirming and progressing false actives. Moreover, in our experience, preparations of purified fulllength and truncated active LRRK2 protein are prone to aggregation, presumably due to the large size of even the truncated proteins, and the fact that LRRK2 is likely to form dimers and complexes in its native cellular state.…”

Lack of Evidence for Decreased Protein Stability in the 2397 (Met) Haplotype of the Leucine Rich Repeat Kinase 2 Protein Implicated in Parkinson’s Disease

“…However, the effects of mutations and polymorphisms in other domains of LRRK2, such as the WD40 domain, are less understood. The WD40 appears to function as a scaffold for protein-protein complexes in the formation of homodimers, for vesicle trafficking, signal transduction, cytoskeleton assembly and autophosphorylation [8]. Therefore, unsurprisingly, several mutations, or deletion, of this domain have been found to contribute to Parkinson's disease [9] [10].…”