Biochemistry and Pharmacology - Research Publications
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ItemThe Roc-COR tandem domain of leucine-rich repeat kinase 2 forms dimers and exhibits conventional Ras-like GTPase properties(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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ItemInsight into the RssB-Mediated Recognition and Delivery of σs to the AAA+ Protease, ClpXP(MDPI, 2020-04)In Escherichia coli, SigmaS (σS) is the master regulator of the general stress response. The cellular levels of σS are controlled by transcription, translation and protein stability. The turnover of σS, by the AAA+ protease (ClpXP), is tightly regulated by a dedicated adaptor protein, termed RssB (Regulator of Sigma S protein B)-which is an atypical member of the response regulator (RR) family. Currently however, the molecular mechanism of σS recognition and delivery by RssB is only poorly understood. Here we describe the crystal structures of both RssB domains (RssBN and RssBC) and the SAXS analysis of full-length RssB (both free and in complex with σS). Together with our biochemical analysis we propose a model for the recognition and delivery of σS by this essential adaptor protein. Similar to most bacterial RRs, the N-terminal domain of RssB (RssBN) comprises a typical mixed (βα)5-fold. Although phosphorylation of RssBN (at Asp58) is essential for high affinity binding of σS, much of the direct binding to σS occurs via the C-terminal effector domain of RssB (RssBC). In contrast to most RRs the effector domain of RssB forms a β-sandwich fold composed of two sheets surrounded by α-helical protrusions and as such, shares structural homology with serine/threonine phosphatases that exhibit a PPM/PP2C fold. Our biochemical data demonstrate that this domain plays a key role in both substrate interaction and docking to the zinc binding domain (ZBD) of ClpX. We propose that RssB docking to the ZBD of ClpX overlaps with the docking site of another regulator of RssB, the anti-adaptor IraD. Hence, we speculate that docking to ClpX may trigger release of its substrate through activation of a "closed" state (as seen in the RssB-IraD complex), thereby coupling adaptor docking (to ClpX) with substrate release. This competitive docking to RssB would prevent futile interaction of ClpX with the IraD-RssB complex (which lacks a substrate). Finally, substrate recognition by RssB appears to be regulated by a key residue (Arg117) within the α5 helix of the N-terminal domain. Importantly, this residue is not directly involved in σS interaction, as σS binding to the R117A mutant can be restored by phosphorylation. Likewise, R117A retains the ability to interact with and activate ClpX for degradation of σS, both in the presence and absence of acetyl phosphate. Therefore, we propose that this region of RssB (the α5 helix) plays a critical role in driving interaction with σS at a distal site.
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ItemManipulating the Lewis antigen specificity of the cholesterol-dependent cytolysin lectinolysin(FRONTIERS MEDIA SA, 2012)The cholesterol-dependent cytolysins (CDCs) attack cells by punching large holes in their membranes. Lectinolysin from Streptococcus mitis is unique among CDCs due to the presence of an N-terminal lectin domain that enhances the pore-forming activity of the toxin. We recently determined the crystal structures of the lectin domain in complex with various glycans. These structures revealed the molecular basis for the Lewis antigen specificity of the toxin. Based on this information we have used in silico molecular modeling to design a mutant toxin, which we predicted would increase its specificity for Lewis y, an antigen found on the surface of cancer cells. Surprisingly, we found by surface plasmon resonance binding experiments that the resultant mutant lectin domain exhibited higher specificity for Lewis b antigens instead. We then undertook comparative crystallographic and molecular dynamics simulation studies of the wild-type and mutant lectin domains to understand the molecular basis for the disparity between the theoretical and experimental results. The crystallographic results revealed that the net number of interactions between Lewis y and wild-type versus mutant was unchanged whereas there was a loss of a hydrogen bond between mutant and Lewis b compared to wild-type. In contrast, the molecular dynamics studies revealed that the Lewis b antigen spent more time in the binding pocket of the mutant compared to wild-type and the reverse was true for Lewis y. The results of these simulation studies are consistent with the conclusions drawn from the surface plasmon resonance studies. This work is part of a program to engineer lectinolysin so that it will target and kill specific cells in human diseases.
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ItemCrystal structure of Streptococcus pneumoniae pneumolysin provides key insights into early steps of pore formation(NATURE PORTFOLIO, 2015-09-25)Pore-forming proteins are weapons often used by bacterial pathogens to breach the membrane barrier of target cells. Despite their critical role in infection important structural aspects of the mechanism of how these proteins assemble into pores remain unknown. Streptococcus pneumoniae is the world's leading cause of pneumonia, meningitis, bacteremia and otitis media. Pneumolysin (PLY) is a major virulence factor of S. pneumoniae and a target for both small molecule drug development and vaccines. PLY is a member of the cholesterol-dependent cytolysins (CDCs), a family of pore-forming toxins that form gigantic pores in cell membranes. Here we present the structure of PLY determined by X-ray crystallography and, in solution, by small-angle X-ray scattering. The crystal structure reveals PLY assembles as a linear oligomer that provides key structural insights into the poorly understood early monomer-monomer interactions of CDCs at the membrane surface.
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ItemSimultaneous Binding of the Anti-Cancer IgM Monoclonal Antibody PAT-SM6 to Low Density Lipoproteins and GRP78(PUBLIC LIBRARY SCIENCE, 2013-04-19)The tumour-derived monoclonal IgM antibody PAT-SM6 specifically kills malignant cells by an apoptotic mechanism linked to the excessive uptake of plasma lipids. The mechanism is postulated to occur via the multi-point attachment of PAT-SM6 to the unfolded protein response regulator GRP78, located on the surface of tumour cells, coupled to the simultaneous binding of plasma low density lipoprotein (LDL). We prepared and characterised LDL and oxidized LDL using sedimentation velocity and small-angle X-ray scattering (SAXS) analysis. Enzyme-linked immunosorbent (ELISA) techniques indicated apparent dissociation constants of approximately 20 nM for the binding of LDL or oxidized LDL to PAT-SM6. ELISA experiments showed cross competition with LDL inhibiting PAT-SM6 binding to immobilised GRP78, while, in the reverse experiment, GRP78 inhibited PAT-SM6 binding to immobilized LDL. In contrast to the results of the ELISA experiments, sedimentation velocity experiments indicated relatively weak interactions between LDL and PAT-SM6, suggesting immunoabsorbance to the microtiter plate is driven by an avidity-based binding mechanism. The importance of avidity and the multipoint attachment of antigens to PAT-SM6 was further investigated using antigen-coated polystyrene beads. Absorption of GRP78 or LDL to polystyrene microspheres led to an increase in the inhibition of PAT-SM6 binding to microtiter plates coated with GRP78 or LDL, respectively. These results support the hypothesis that the biological action of PAT-SM6 in tumour cell apoptosis depends on the multivalent nature of PAT-SM6 and the ability to interact simultaneously with LDL and multiple GRP78 molecules clustered on the tumour cell surface.
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ItemThe Anti-Cancer IgM Monoclonal Antibody PAT-SM6 Binds with High Avidity to the Unfolded Protein Response Regulator GRP78(PUBLIC LIBRARY SCIENCE, 2012-09-19)The monoclonal IgM antibody PAT-SM6 derived from human tumours induces apoptosis in tumour cells and is considered a potential anti-cancer agent. A primary target for PAT-SM6 is the unfolded protein response regulator GRP78, over-expressed externally on the cell surface of tumour cells. Small angle X-ray scattering (SAXS) studies of human GRP78 showed a two-domain dumbbell-shaped monomer, while SAXS analysis of PAT-SM6 revealed a saucer-shaped structure accommodating five-fold symmetry, consistent with previous studies of related proteins. Sedimentation velocity analysis of GRP78 and PAT-SM6 mixtures indicated weak complex formation characterized by dissociation constants in the high micromolar concentration range. In contrast, enzyme-linked immunosorbant assays (ELISAs) showed strong and specific interactions between PAT-SM6 and immobilized GRP78. The apparent binding constant estimated from a PAT-SM6 saturation curve correlated strongly with the concentration of GRP78 used to coat the microtiter tray. Experiments using polyclonal antiGRP78 IgG antibodies or a monoclonal IgG derivative of PAT-SM6 did not show a similar dependence. Competition experiments with soluble GRP78 indicated more effective inhibition of PAT-SM6 binding at low GRP78 coating concentrations. These observations suggest an avidity-based binding mechanism that depends on the multi-point attachment of PAT-SM6 to GRP78 clustered on the surface of the tray. Analysis of ELISA data at high GRP78 coating concentrations yielded an apparent dissociation constant of approximately 4 nM. We propose that the biological action of PAT-SM6 in tumour cell apoptosis may depend on the multivalent nature of PAT-SM6 and the high avidity of its interaction with multiple GRP78 molecules clustered on the tumour cell surface.
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ItemA Biosensor of Src Family Kinase Conformation by Exposable Tetracysteine Useful for Cell-Based Screening(AMER CHEMICAL SOC, 2014-07)We developed a new approach to distinguish distinct protein conformations in live cells. The method, exposable tetracysteine (XTC), involved placing an engineered tetracysteine motif into a target protein that has conditional access to biarsenical dye binding by conformational state. XTC was used to distinguish open and closed regulatory conformations of Src family kinases. Substituting just four residues with cysteines in the conserved SH2 domain of three Src-family kinases (c-Src, Lck, Lyn) enabled open and closed conformations to be monitored on the basis of binding differences to biarsenical dyes FlAsH or ReAsH. Fusion of the kinases with a fluorescent protein tracked the kinase presence, and the XTC approach enabled simultaneous assessment of regulatory state. The c-Src XTC biosensor was applied in a boutique screen of kinase inhibitors, which revealed six compounds to induce conformational closure. The XTC approach demonstrates new potential for assays targeting conformational changes in key proteins in disease and biology.
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ItemPrediction 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)(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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ItemTyrosine 416 Is Phosphorylated in the Closed, Repressed Conformation of c-Src(PUBLIC LIBRARY SCIENCE, 2013-07-26)c-Src kinase activity is regulated by phosphorylation of Y527 and Y416. Y527 phosphorylation stabilizes a closed conformation, which suppresses kinase activity towards substrates, whereas phosphorylation at Y416 promotes an elevated kinase activity by stabilizing the activation loop in a manner permissive for substrate binding. Here we investigated the correlation of Y416 phosphorylation with c-Src activity when c-Src was locked into the open and closed conformations (by mutations Y527F and Q528E, P529E, G530I respectively). Consistent with prior findings, we found Y416 to be more greatly phosphorylated when c-Src was in an open, active conformation. However, we also observed an appreciable amount of Y416 was phosphorylated when c-Src was in a closed, repressed conformation under conditions by which c-Src was unable to phosphorylate substrate STAT3. The phosphorylation of Y416 in the closed conformation arose by autophosphorylation, since abolishing kinase activity by mutating the ATP binding site (K295M) prevented phosphorylation. Basal Y416 phosphorylation correlated positively with cellular levels of c-Src suggesting autophosphorylation depended on self-association. Using sedimentation velocity analysis on cell lysate with fluorescence detection optics, we confirmed that c-Src forms monomers and dimers, with the open conformation also forming a minor population of larger mass complexes. Collectively, our studies suggest a model by which dimerization of c-Src primes c-Src via Y416 phosphorylation to enable rapid potentiation of activity when Src adopts an open conformation. Once in the open conformation, c-Src can amplify the response by recruiting and phosphorylating substrates such as STAT3 and increasing the extent of autophosphorylation.