Biochemistry and Pharmacology - Research Publications

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    The Roc-COR tandem domain of leucine-rich repeat kinase 2 forms dimers and exhibits conventional Ras-like GTPase properties
    Mills, RD ; Liang, L-Y ; Lio, DS-S ; Mok, Y-F ; Mulhern, TD ; Cao, G ; Griffin, M ; Kenche, VB ; Culvenor, JG ; Cheng, H-C (WILEY, 2018-11)
    The Parkinson's disease (PD)-causative leucine-rich repeat kinase 2 (LRRK2) belongs to the Roco family of G-proteins comprising a Ras-of-complex (Roc) domain followed by a C-terminal of Roc (COR) domain in tandem (called Roc-COR domain). Two prokaryotic Roc-COR domains have been characterized as 'G proteins activated by guanine nucleotide-dependent dimerization' (GADs), which require dimerization for activation of their GTPase activity and bind guanine nucleotides with relatively low affinities. Additionally, LRRK2 Roc domain in isolation binds guanine nucleotides with relatively low affinities. As such, LRRK2 GTPase domain was predicted to be a GAD. Herein, we describe the design and high-level expression of human LRRK2 Roc-COR domain (LRRK2 Roc-COR). Biochemical analyses of LRRK2 Roc-COR reveal that it forms homodimers, with the C-terminal portion of COR mediating its dimerization. Furthermore, it co-purifies and binds Mg2+ GTP/GDP at 1 : 1 stoichiometry, and it hydrolyzes GTP with Km  and kcat  of 22 nM and 4.70 × 10-4  min-1 ,  respectively. Thus, even though LRRK2 Roc-COR forms GAD-like homodimers, it exhibits conventional Ras-like GTPase properties, with high-affinity binding of Mg2+ -GTP/GDP and low intrinsic catalytic activity. The PD-causative Y1699C mutation mapped to the COR domain was previously reported to reduce the GTPase activity of full-length LRRK2. In contrast, this mutation induces no change in the GTPase activity, and only slight perturbations in the secondary structure contents of LRRK2 Roc-COR. As this mutation does not directly affect the GTPase activity of the isolated Roc-COR tandem, it is possible that the effects of this mutation on full-length LRRK2 occur via other functional domains. Open Practices Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/.
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    Analysis of LRRK2 accessory repeat domains: prediction of repeat length, number and sites of Parkinson's disease mutations
    MILLS, R ; Mulhern, TD ; Cheng, HC ; Culvenor, JG ( 2012)
    Various investigators have identified the major domain organization of LRRK2 (leucine-rich repeat kinase 2), which includes a GTPase ROC (Ras of complex proteins) domain followed by a COR (C-terminal of ROC) domain and a protein kinase domain. In addition, there are four domains composed of structural repeat motifs likely to be involved in regulation and localization of this complex protein. In the present paper, we report our bioinformatic analyses of the human LRRK2 amino acid sequence to predict the repeat size, number and likely boundaries for the armadillo repeat, ankyrin repeat, the leucine-rich repeat and WD40 repeat regions of LRRK2. Homology modelling using known protein structures with similar domains was used to predict structures, exposed residues and location of mutations for these repeat regions. We predict that the armadillo repeats, ankyrin repeats and leucine-rich repeats together form an extended N-terminal flexible 'solenoid'-like structure composed of tandem repeat modules likely to be important in anchoring to the membrane and cytoskeletal structures as well as binding to other protein ligands. Near the C-terminus of LRRK2, the WD40 repeat region is predicted to form a closed propeller structure that is important for protein complex formation.
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    Prediction of the Repeat Domain Structures and Impact of Parkinsonism-Associated Variations on Structure and Function of all Functional Domains of Leucine-Rich Repeat Kinase 2 (LRRK2)
    Mills, RD ; Mulhern, TD ; Liu, F ; Culvenor, JG ; Cheng, H-C (WILEY, 2014-04)
    Genetic variations of leucine-rich repeat kinase 2 (LRRK2) are the major cause of dominantly inherited Parkinson disease (PD). LRRK2 protein contains seven predicted domains: a tandem Ras-like GTPase (ROC) domain and C-terminal of Roc (COR) domain, a protein kinase domain, and four repeat domains. PD-causative variations arise in all domains, suggesting that aberrant functioning of any domain can contribute to neurotoxic mechanisms of LRRK2. Determination of the three-dimensional structure of LRRK2 is one of the best avenues to decipher its neurotoxic mechanism. However, with the exception of the Roc domain, the three-dimensional structures of the functional domains of LRRK2 have yet to be determined. Based on the known three-dimensional structures of repeat domains of other proteins, the tandem Roc-COR domains of the Chlorobium tepidum Rab family protein, and the kinase domain of the Dictyostelium discoideum Roco4 protein, we predicted (1) the motifs essential for protein-protein interactions in all domains, (2) the motifs critical for catalysis and substrate recognition in the tandem Roc-COR and kinase domains, and (3) the effects of some PD-associated missense variations on the neurotoxic action of LRRK2. Results of our analysis provide a conceptual framework for future investigation into the regulation and the neurotoxic mechanism of LRRK2.
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    Analysis of the Regulatory and Catalytic Domains of PTEN-Induced Kinase-1 (PINK1)
    Sim, CH ; Gabriel, K ; Mills, RD ; Culvenor, JG ; Cheng, H-C (WILEY, 2012-10)
    Mutations of the phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1) gene can cause early-onset familial Parkinson disease (PD). PINK1 encodes a neuroprotective protein kinase localized at the mitochondria, and its involvement in regulating mitochondrial dynamics, trafficking, structure, and function is well documented. Owing to the lack of information on structure and biochemical properties for PINK1, exactly how PINK1 exerts its neuroprotective function and how the PD-causative mutations impact on PINK1 structure and function remain unclear. As an approach to address these questions, we conducted bioinformatic analyses of the mitochondrial targeting, the transmembrane, and kinase domains of PINK1 to predict the motifs governing its regulation and function. Our report sheds light on how PINK1 is targeted to the mitochondria and how PINK1 is cleaved by mitochondrial peptidases. Moreover, it includes a potential optimal phosphorylation sequence preferred by the PINK1 kinase domain. On the basis of the results of our analyses, we predict how the PD-causative mutations affect processing of PINK1 in the mitochondria, PINK1 kinase activity, and substrate specificity. In summary, our results provide a conceptual framework for future investigation of the structural and biochemical basis of regulation and the neuroprotective mechanism of PINK1.