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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ItemPolymorphism in disease-related apolipoprotein C-II amyloid fibrils: a structural model for rod-like fibrils(WILEY, 2018-08)Human apolipoprotein (apo) C-II is one of several plasma apolipoproteins that form amyloid deposits in vivo and is an independent risk factor for cardiovascular disease. Lipid-free apoC-II readily self-assembles into twisted-ribbon amyloid fibrils but forms straight, rod-like amyloid fibrils in the presence of low concentrations of micellar phospholipids. Charge mutations exerted significantly different effects on rod-like fibril formation compared to their effects on twisted-ribbon fibril formation. For instance, the double mutant, K30D-D69K apoC-II, readily formed twisted-ribbon fibrils, while the rate of rod-like fibril formation in the presence of micellar phospholipid was negligible. Structural analysis of rod-like apoC-II fibrils, using hydrogen-deuterium exchange and NMR analysis showed exchange protection consistent with a core cross-β structure comprising the C-terminal 58-76 region. Molecular dynamics simulations of fibril arrangements for this region favoured a parallel cross-β structure. X-ray fibre diffraction data for aligned rod-like fibrils showed a major meridional spacing at 4.6 Å and equatorial spacings at 9.7, 23.8 and 46.6 Å. The latter two equatorial spacings are not observed for aligned twisted-ribbon fibrils and are predicted for a model involving two cross-β fibrils in an off-set antiparallel structure with four apoC-II units per rise of the β-sheet. This model is consistent with the mutational effects on rod-like apoC-II fibril formation. The lipid-dependent polymorphisms exhibited by apoC-II fibrils could determine the properties of apoC-II in renal amyloid deposits and their potential role in the development of cardiovascular disease.
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ItemCrystal structure of TcpK in complex with oriT DNA of the antibiotic resistance plasmid pCW3(NATURE PUBLISHING GROUP, 2018-09-13)Conjugation is fundamental for the acquisition of new genetic traits and the development of antibiotic resistance in pathogenic organisms. Here, we show that a hypothetical Clostridium perfringens protein, TcpK, which is encoded by the tetracycline resistance plasmid pCW3, is essential for efficient conjugative DNA transfer. Our studies reveal that TcpK is a member of the winged helix-turn-helix (wHTH) transcription factor superfamily and that it forms a dimer in solution. Furthermore, TcpK specifically binds to a nine-nucleotide sequence that is present as tandem repeats within the pCW3 origin of transfer (oriT). The X-ray crystal structure of the TcpK-TcpK box complex reveals a binding mode centered on and around the β-wing, which is different from what has been previously shown for other wHTH proteins. Structure-guided mutagenesis experiments validate the specific interaction between TcpK and the DNA molecule. Additional studies highlight that the TcpK dimer is important for specific DNA binding.
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ItemNo Preview AvailableA Multilaboratory Comparison of Calibration Accuracy and the Performance of External References in Analytical Ultracentrifugation(PUBLIC LIBRARY SCIENCE, 2015-05-21)Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.
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ItemA Cyclic Peptide Inhibitor of ApoC-II Peptide Fibril Formation: Mechanistic Insight from NMR and Molecular Dynamics Analysis(ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD, 2012-03-09)The misfolding and aggregation of proteins to form amyloid fibrils is a characteristic feature of several common age-related diseases. Agents that directly inhibit formation of amyloid fibrils represent one approach to combating these diseases. We have investigated the potential of a cyclic peptide to inhibit fibril formation by fibrillogenic peptides from human apolipoprotein C-II (apoC-II). Cyc[60-70] was formed by disulfide cross-linking of cysteine residues added to the termini of the fibrillogenic peptide comprising apoC-II residues 60-70. This cyclic peptide did not self-associate into fibrils. However, substoichiometric concentrations of cyc[60-70] significantly delayed fibril formation by the fibrillogenic, linear peptides apoC-II[60-70] and apoC-II[56-76]. Reduction of the disulfide bond or scrambling the amino acid sequence within cyc[60-70] significantly impaired its inhibitory activity. The solution structure of cyc[60-70] was solved using NMR spectroscopy, revealing a well-defined structure comprising a hydrophilic face and a more hydrophobic face containing the Met60, Tyr63, Ile66 and Phe67 side chains. Molecular dynamics (MD) studies identified a flexible central region within cyc[60-70], while MD simulations of "scrambled" cyc[60-70] indicated an increased formation of intramolecular hydrogen bonds and a reduction in the overall flexibility of the peptide. Our structural studies suggest that the inhibitory activity of cyc[60-70] is mediated by an elongated structure with inherent flexibility and distinct hydrophobic and hydrophilic faces, enabling cyc[60-70] to interact transiently with fibrillogenic peptides and inhibit fibril assembly. These results suggest that cyclic peptides based on amyloidogenic core peptides could be useful as specific inhibitors of amyloid fibril formation.
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ItemSize analysis of polyglutamine protein aggregates using fluorescence detection in an analytical ultracentrifuge.(Springer Nature, 2013)Defining the aggregation process of proteins formed by poly-amino acid repeats in cells remains a challenging task due to a lack of robust techniques for their isolation and quantitation. Sedimentation velocity methodology using fluorescence detected analytical ultracentrifugation is one approach that can offer significant insight into aggregation formation and kinetics. While this technique has traditionally been used with purified proteins, it is now possible for substantial information to be collected with studies using cell lysates expressing a GFP-tagged protein of interest. In this chapter, we describe protocols for sample preparation and setting up the fluorescence detection system in an analytical ultracentrifuge to perform sedimentation velocity experiments on cell lysates containing aggregates formed by poly-amino acid repeat proteins.
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ItemNo Preview AvailableThe Allosteric Mechanism Induced by Protein Kinase A (PKA) Phosphorylation of Dematin (Band 4.9)(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2013-03-22)Dematin (band 4.9) is an F-actin binding and bundling protein best known for its role within red blood cells, where it both stabilizes as well as attaches the spectrin/actin cytoskeleton to the erythrocytic membrane. Here, we investigate the structural consequences of phosphorylating serine 381, a covalent modification that turns off F-actin bundling activity. In contrast to the canonical doctrine, in which phosphorylation of an intrinsically disordered region/protein confers affinity for another domain/protein, we found the converse to be true of dematin: phosphorylation of the well folded C-terminal villin-type headpiece confers affinity for its intrinsically disordered N-terminal core domain. We employed analytical ultracentrifugation to demonstrate that dematin is monomeric, in contrast to the prevailing view that it is trimeric. Next, using a series of truncation mutants, we verified that dematin has two F-actin binding sites, one in the core domain and the other in the headpiece domain. Although the phosphorylation-mimicking mutant, S381E, was incapable of bundling microfilaments, it retains the ability to bind F-actin. We found that a phosphorylation-mimicking mutant, S381E, eliminated the ability to bundle, but not bind F-actin filaments. Lastly, we show that the S381E point mutant caused the headpiece domain to associate with the core domain, leading us to the mechanism for cAMP-dependent kinase control of dematin's F-actin bundling activity: when unphosphorylated, dematin's two F-actin binding domains move independent of one another permitting them to bind different F-actin filaments. Phosphorylation causes these two domains to associate, forming a compact structure, and sterically eliminating one of these F-actin binding sites.
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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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ItemNo Preview AvailableAMP-Activated Protein Kinase β-Subunit Requires Internal Motion for Optimal Carbohydrate Binding(CELL PRESS, 2012-01-18)AMP-activated protein kinase interacts with oligosaccharides and glycogen through the carbohydrate-binding module (CBM) containing the β-subunit, for which there are two isoforms (β(1) and β(2)). Muscle-specific β(2)-CBM, either as an isolated domain or in the intact enzyme, binds carbohydrates more tightly than the ubiquitous β(1)-CBM. Although residues that contact carbohydrate are strictly conserved, an additional threonine in a loop of β(2)-CBM is concurrent with an increase in flexibility in β(2)-CBM, which may account for the affinity differences between the two isoforms. In contrast to β(1)-CBM, unbound β(2)-CBM showed microsecond-to-millisecond motion at the base of a β-hairpin that contains residues that make critical contacts with carbohydrate. Upon binding to carbohydrate, similar microsecond-to-millisecond motion was observed in this β-hairpin and the loop that contains the threonine insertion. Deletion of the threonine from β(2)-CBM resulted in reduced carbohydrate affinity. Although motion was retained in the unbound state, a significant loss of motion was observed in the bound state of the β(2)-CBM mutant. Insertion of a threonine into the background of β(1)-CBM resulted in increased ligand affinity and flexibility in these loops when bound to carbohydrate. However, these mutations indicate that the additional threonine is not solely responsible for the differences in carbohydrate affinity and protein dynamics. Nevertheless, these results suggest that altered protein dynamics may contribute to differences in the ligand affinity of the two naturally occurring CBM isoforms.
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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.