Structure of the Roc–COR domain tandem of C. tepidum, a prokaryotic homologue of the human LRRK2 Parkinson kinase
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ABSTRACT:
SUBMITTER: Gotthardt K
PROVIDER: S-EPMC2529373 | biostudies-literature | 2008 Sep
REPOSITORIES: biostudies-literature
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Project description:Ras of complex proteins (Roc) belongs to the superfamily of Ras-related small G-proteins that always occurs in tandem with the C-terminal of Roc (COR) domain. This Roc-COR tandem is found in the bacterial and eukaryotic world. Its most prominent member is the leucine-rich repeat kinase LRRK2, which is mutated and activated in Parkinson patients. Here, we investigated biochemically and structurally the Roco protein from Chlorobium tepidum. We show that Roc is highly homologous to Ras, whereas the COR domain is a dimerisation device. The juxtaposition of the G-domains and mutational analysis suggest that the Roc GTPase reaction is stimulated and/or regulated by dimerisation in a nucleotide-dependent manner. The region most conserved between bacteria and man is the interface between Roc and COR, where single-point Parkinson mutations of the Roc and COR domains are in close proximity. The analogous mutations in C. tepidum Roc-COR decrease the GTPase reaction rate, most likely due to a modification of the interaction between the Roc and COR domains.
| S-EPMC2519104 | biostudies-literature
Project description:Ras of complex proteins (Roc) is a Ras-like GTP-binding domain that always occurs in tandem with the C-terminal of Roc (COR) domain and is found in bacteria, plants and animals. Recently, it has been shown that Roco proteins belong to the family of G-proteins activated by nucleotide (nt)-dependent dimerization (GADs). We investigated the RocCOR tandem from the bacteria Chlorobium tepidum with site-directed spin labelling and pulse EPR distance measurements to follow conformational changes during the Roco G-protein cycle. Our results confirm that the COR domains are a stable dimerization device serving as a scaffold for the Roc domains that, in contrast, are structurally heterogeneous and dynamic entities. Contrary to other GAD proteins, we observed only minor structural alterations upon binding and hydrolysis of GTP, indicating significant mechanistic variations within this protein class. Mutations in the most prominent member of the Roco family of proteins, leucine-rich repeat (LRR) kinase 2 (LRRK2), are the most frequent cause of late-onset Parkinson's disease (PD). Using a stable recombinant LRRK2 Roc-COR-kinase fragment we obtained detailed kinetic data for the G-protein cycle. Our data confirmed that dimerization is essential for efficient GTP hydrolysis and PD mutations in the Roc domain result in decreased GTPase activity. Previous data have shown that these LRRK2 PD-mutations are located in the interface between Roc and COR. Importantly, analogous mutations in the conserved C. tepidum Roc/COR interface significantly influence the structure and nt-induced conformational changes of the Roc domains.
| S-EPMC4721548 | biostudies-literature
Project description:Mutations in PARK8, encoding LRRK2, are the most common known cause of Parkinson's disease. The LRRK2 Roc-COR tandem domain exhibits GTPase activity controlling LRRK2 kinase activity via an intramolecular process. We report the interaction of LRRK2 with the dishevelled family of phosphoproteins (DVL1-3), key regulators of Wnt (Wingless/Int) signalling pathways important for axon guidance, synapse formation and neuronal maintenance. Interestingly, DVLs can interact with and mediate the activation of small GTPases with structural similarity to the LRRK2 Roc domain. The LRRK2 Roc-COR domain and the DVL1 DEP domain were necessary and sufficient for LRRK2-DVL1 interaction. Co-expression of DVL1 increased LRRK2 steady-state protein levels, an effect that was dependent on the DEP domain. Strikingly, LRRK2-DVL1-3 interactions were disrupted by the familial PARK8 mutation Y1699C, whereas pathogenic mutations at residues R1441 and R1728 strengthened LRRK2-DVL1 interactions. Co-expression of DVL1 with LRRK2 in mammalian cells resulted in the redistribution of LRRK2 to typical cytoplasmic DVL1 aggregates in HEK293 and SH-SY5Y cells and co-localization in neurites and growth cones of differentiated dopaminergic SH-SY5Y cells. This is the first report of the modulation of a key LRRK2-accessory protein interaction by PARK8 Roc-COR domain mutations segregating with Parkinson's disease. Since the DVL1 DEP domain is known to be involved in the regulation of small GTPases, we propose that: (i) DVLs may influence LRRK2 GTPase activity, and (ii) Roc-COR domain mutations modulating LRRK2-DVL interactions indirectly influence kinase activity. Our findings also link LRRK2 to Wnt signalling pathways, suggesting novel pathogenic mechanisms and new targets for genetic analysis in Parkinson's disease.
| S-EPMC2748899 | biostudies-other
Project description:Leucine-rich repeat kinase 2 (LRRK2) mutations are the most common genetic cause of Parkinson's disease. LRRK2 is a multifunctional protein affecting many cellular processes and has been described to bind microtubules. Defective microtubule-based axonal transport is hypothesized to contribute to Parkinson's disease, but whether LRRK2 mutations affect this process to mediate pathogenesis is not known. Here we find that LRRK2 containing pathogenic Roc-COR domain mutations (R1441C, Y1699C) preferentially associates with deacetylated microtubules, and inhibits axonal transport in primary neurons and in Drosophila, causing locomotor deficits in vivo. In vitro, increasing microtubule acetylation using deacetylase inhibitors or the tubulin acetylase αTAT1 prevents association of mutant LRRK2 with microtubules, and the deacetylase inhibitor trichostatin A (TSA) restores axonal transport. In vivo knockdown of the deacetylases HDAC6 and Sirt2, or administration of TSA rescues both axonal transport and locomotor behavior. Thus, this study reveals a pathogenic mechanism and a potential intervention for Parkinson's disease.
| S-EPMC4208097 | biostudies-literature
Project description:Roco proteins are characterized by the presence of a Roc-COR supradomain harbouring GTPase activity, which is often preceded by an LRR domain. The most notorious member of the Roco protein family is the Parkinson's disease-associated LRRK2. The Roco protein from the bacterium Chlorobium tepidum has been used as a model system to investigate the structure and mechanism of this class of enzymes. Here, the crystallization and crystallographic analysis of the LRR-Roc-COR construct of the C. tepidum Roco protein is reported. The LRR-Roc-COR crystals belonged to space group P212121, with unit-cell parameters a = 95.6, b = 129.8, c = 179.5?Å, ? = ? = ? = 90°, and diffracted to a resolution of 3.3?Å. Based on the calculated Matthews coefficient, Patterson map analysis and an initial molecular-replacement analysis, one protein dimer is present in the asymmetric unit. The crystal structure of this protein will provide valuable insights into the interaction between the Roc-COR and LRR domains within Roco proteins.
| S-EPMC5619744 | biostudies-literature
Project description:BACKGROUND:Recent studies have linked certain single nucleotide polymorphisms in the leucine-rich repeat kinase 2 (LRRK2) gene with Parkinson's disease (PD). Among the mutations, LRRK2 c.4883G>C (R1628P) variant was identified to have a significant association with the risk of PD in ethnic Han-Chinese populations. But the molecular pathological mechanisms of R1628P mutation in PD is still unknown. PRINCIPLE FINDINGS:Unlike other LRRK2 mutants in the Roc-COR-Kinase domain, the R1628P mutation didn't alter the LRRK2 kinase activity and promote neuronal death directly. LRRK2 R1628P mutation increased the binding affinity of LRRK2 with Cyclin-dependent kinase 5 (Cdk5). Interestingly, R1628P mutation turned its adjacent amino acid residue S1627 on LRRK2 protein to a novel phosphorylation site of Cdk5, which could be defined as a typical type II (+) phosphorylation-related single nucleotide polymorphism. Importantly, we showed that the phosphorylation of S1627 by Cdk5 could activate the LRRK2 kinase, and neurons ectopically expressing R1628P displayed a higher sensitivity to 1-methyl-4-phenylpyridinium, a bioactive metabolite of environmental toxin MPTP, in a Cdk5-dependent manner. CONCLUSION:Our data indicate that Parkinson-related LRRK2 mutation R1628P leads to Cdk5 phosphorylation of LRRK2 at S1627, which would upregulate the kinase activity of LRRK2 and consequently cause neuronal death.
| S-EPMC4773127 | biostudies-literature
Project description:LRRK2 mutations are recognized as the most frequent genetic cause of both familial and sporadic parkinsonism identified to date. A remarkable feature of this form of parkinsonism is the variable penetrance of symptom manifestation resulting in a wide range of age-at-onset in patients. Herein we use a functional approach to identify the Lrrk1 protein as a potential disease modifier demonstrating an interaction and heterodimer formation with Lrrk2. In addition, evaluation of LRRK1 variants in our large Lrrk2 p.G2019S-parkinsonism series from a Tunisian (n=145) identified a missense mutation (p.L416M) resulting in an average 6.2 years younger age at disease onset. In conclusion we show that the interaction of Lrrk1-Lrrk2 can form protein dimers and this interaction may influence the age of symptomatic manifestation in Lrrk2-parkinsonism patients.
| S-EPMC2847049 | biostudies-literature
Project description:Mutation in leucine-rich-repeat kinase 2 (LRRK2) is a common cause of Parkinson disease (PD). A disease-causing point mutation R1441H/G/C in the GTPase domain of LRRK2 leads to overactivation of its kinase domain. However, the mechanism by which this mutation alters the normal function of its GTPase domain [Ras of complex proteins (Roc)] remains unclear. Here, we report the effects of R1441H mutation (RocR1441H) on the structure and activity of Roc. We show that Roc forms a stable monomeric conformation in solution that is catalytically active, thus demonstrating that LRRK2 is a bona fide self-contained GTPase. We further show that the R1441H mutation causes a twofold reduction in GTPase activity without affecting the structure, thermal stability, and GDP-binding affinity of Roc. However, the mutation causes a twofold increase in GTP-binding affinity of Roc, thus suggesting that the PD-causing mutation R1441H traps Roc in a more persistently activated state by increasing its affinity for GTP and, at the same time, compromising its GTP hydrolysis.
| S-EPMC3964117 | biostudies-literature
Project description:Protein kinases and GTPases are the two major molecular switches that regulate much of biology, and both of these domains are embedded within the large multi-domain Leucine-Rich Repeat Kinase 2 (LRRK2). Mutations in LRRK2 are the most common cause of familial Parkinson's disease (PD) and are also implicated in Crohn's disease. The recent Cryo-Electron Microscopy (Cryo-EM) structure of the four C-terminal domains [ROC COR KIN WD40 (RCKW)] of LRRK2 includes both of the catalytic domains. Although the important allosteric N-terminal domains are missing in the Cryo-EM structure this structure allows us to not only explore the conserved features of the kinase domain, which is trapped in an inactive and open conformation but also to observe the direct allosteric cross-talk between the two domains. To define the unique features of the kinase domain and to better understand the dynamic switch mechanism that allows LRRK2 to toggle between its inactive and active conformations, we have compared the LRRK2 kinase domain to Src, BRaf, and PKA. We also compare and contrast the two canonical glycine-rich loop motifs in LRRK2 that anchor the nucleotide: the G-Loop in protein kinases that anchors ATP and the P-Loop in GTPases that anchors GTP. The RCKW structure also provides a template for the cross-talk between the kinase and GTPase domains and brings new mechanistic insights into the physiological function of LRRK2 and how the kinase domain, along with key phosphorylation sites, can serve as an allosteric hub for mediating conformational changes.
| S-EPMC7573214 | biostudies-literature
Project description:Mutations in leucine-rich repeat kinase 2 (LRRK2) are recognized as the most frequent cause of Parkinson's disease (PD). As a multidomain ROCO protein, LRRK2 is characterized by the presence of both a Ras-of-complex (ROC) GTPase domain and a kinase domain connected through the C-terminal of an ROC domain (COR). The bienzymatic ROC-COR-kinase catalytic triad indicated the potential role of GTPase domain in regulating kinase activity. However, as a functional GTPase, the detailed intrinsic regulation of the ROC activation cycle remains poorly understood. Here, combining extensive molecular dynamics simulations and Markov state models, we disclosed the dynamic structural rearrangement of ROC's homodimer during nucleotide turnover. Our study revealed the coupling between dimerization extent and nucleotide-binding state, indicating a nucleotide-dependent dimerization-based activation scheme adopted by ROC GTPase. Furthermore, inspired by the well-known R1441C/G/H PD-relevant mutations within the ROC domain, we illuminated the potential allosteric molecular mechanism for its pathogenetic effects through enabling faster interconversion between inactive and active states, thus trapping ROC in a prolonged activated state, while the implicated allostery could provide further guidance for identification of regulatory allosteric pockets on the ROC complex. Our investigations illuminated the thermodynamics and kinetics of ROC homodimer during nucleotide-dependent activation for the first time and provided guidance for further exploiting ROC as therapeutic targets for controlling LRRK2 functionality in PD treatment.
| S-EPMC8467336 | biostudies-literature