ORCID Profile
0000-0001-5452-5193
Current Organisation
CSIRO
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In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Pharmacology and Pharmaceutical Sciences | Medical Biochemistry: Proteins And Peptides | Biochemistry And Cell Biology Not Elsewhere Classified | Biochemistry and Cell Biology | Pharmaceutical Sciences And Pharmacy | Biomedical Engineering | Biomaterials | Enzymes | Medical Biotechnology | Structural Biology (incl. Macromolecular Modelling) |
Treatments (e.g. chemicals, antibiotics) | Respiratory system and diseases (incl. asthma) | Expanding Knowledge in the Biological Sciences | Biological sciences | Inherited diseases (incl. gene therapy)
Publisher: CSIRO Publishing
Date: 2020
DOI: 10.1071/CH19346
Abstract: Superoxide dismutase (SOD) is an almost ubiquitous metalloenzyme in aerobic organisms that catalyses the disproportionation of superoxide. Geobacillus stearothermophilus MnSOD is the only published MnSOD structure that does not have its coordinates publicly available, yet it is one of the more cited structures in the SOD literature. The structure has now been refined with modern programs, yielding a significantly improved structure which has been deposited in the Protein Data Bank. Importantly, the further refined structure reveals the presence of a catalytically important fifth ligand, water, to the metal centre, as observed in other SOD structures.
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 13-07-2010
Abstract: Inhibitors of insulin-regulated aminopeptidase (IRAP) improve memory and are being developed as a novel treatment for memory loss. In this study, the binding of a class of these inhibitors to human IRAP was investigated using molecular docking and site-directed mutagenesis. Four benzopyran-based IRAP inhibitors with different affinities were docked into a homology model of the catalytic site of IRAP. Two 4-pyridinyl derivatives orient with the benzopyran oxygen interacting with the Zn(2+) ion and a direct parallel ring-stack interaction between the benzopyran rings and Phe544. In contrast, the two 4-quinolinyl derivatives orient in a different manner, interacting with the Zn(2+) ion via the quinoline nitrogen, and Phe544 contributes an edge-face hydrophobic stacking point with the benzopyran moiety. Mutagenic replacement of Phe544 with alanine, isoleucine, or valine resulted in either complete loss of catalytic activity or altered hydrolysis velocity that was substrate-dependent. Phe544 is also important for inhibitor binding, because these mutations altered the K(i) in some cases, and docking of the inhibitors into the corresponding Phe544 mutant models revealed how the interaction might be disturbed. These findings demonstrate a key role of Phe544 in the binding of the benzopyran IRAP inhibitors and for optimal positioning of enzyme substrates during catalysis.
Publisher: Elsevier BV
Date: 07-2003
DOI: 10.1016/S0042-6822(03)00115-6
Abstract: Respiratory syncytial virus (RSV) is a ubiquitous human pathogen and the leading cause of lower respiratory tract infections in infants. Infection of cells and subsequent formation of syncytia occur through membrane fusion mediated by the RSV fusion protein (RSV-F). A novel in vitro assay of recombinant RSV-F function has been devised and used to characterize a number of escape mutants for three known inhibitors of RSV-F that have been isolated. Homology modeling of the RSV-F structure has been carried out on the basis of a chimera derived from the crystal structures of the RSV-F core and a fragment from the orthologous fusion protein from Newcastle disease virus (NDV). The structure correlates well with the appearance of RSV-F in electron micrographs, and the residues identified as contributing to specific binding sites for several monoclonal antibodies are arranged in appropriate solvent-accessible clusters. The positions of the characterized resistance mutants in the model structure identify two promising regions for the design of fusion inhibitors.
Publisher: Research Square Platform LLC
Date: 27-07-2023
DOI: 10.21203/RS.3.RS-3198291/V1
Abstract: Malaria poses an enormous threat to human health. With ever increasing resistance to currently deployed drugs, breakthrough compounds with novel mechanisms of action are urgently needed. Here, we explore pyrimidine-based sulfonamides as a new low molecular weight inhibitor class with drug-like physical parameters and a synthetically accessible scaffold. We show that the exemplar, OSM-S-106, has potent activity against parasite cultures, low mammalian cell toxicity and low propensity for resistance development. In vitro evolution of resistance using a slow r -up approach pointed to the Plasmodium falciparum cytoplasmic asparaginyl tRNA synthetase (PfAsnRS) as the target, consistent with our finding that OSM-S-106 inhibits protein translation and activates the amino acid starvation response. Targeted mass spectrometry confirms that OSM-S-106 is a pro-inhibitor and that inhibition of PfAsnRS occurs via enzyme-mediated production of an Asn-OSM-S-106 adduct. Human AsnRS is much less susceptible to this reaction hijacking mechanism. X-ray crystallographic studies of human AsnRS in complex with inhibitor adducts and docking of pro-inhibitors into a model of Asn-tRNA-bound PfAsnRS provide insights into the structure activity relationship and the selectivity mechanism.
Publisher: Elsevier
Date: 2021
Publisher: Springer Science and Business Media LLC
Date: 1997
Publisher: Wiley
Date: 09-1993
Abstract: Two murine interleukin-6 (mIL-6) variants were constructed using the polymerase chain reaction (PCR), one lacking the last five residues (183-187) at the C-terminus (pMC5) and another with the last five residues of mIL-6 substituted by the corresponding residues of human IL-6 (pMC5H). The growth stimulatory activity of pMC5 on the mouse hybridoma cell line 7TD1 was < 0.05% of mIL-6, whereas pMC5H and mIL-6 were equipotent. The loss of biological activity of pMC5 correlated with its negligible receptor binding affinity on 7TD1 cells, while the binding of pMC5H was comparable to that of mIL-6. Both pMC5 and pMC5H, like mIL-6, failed to interact with recombinant soluble human IL-6 receptor when assayed by surface plasmon resonance-based biosensor analysis. These studies suggest that the C-terminal seven amino acids of human IL-6, alone, do not define species specificity for receptor binding. A variety of biophysical techniques, as well as the binding of a conformational-specific monoclonal antibody, indicated that the global fold of the mIL-6 variants was similar to that of mIL-6, although small changes in the NMR spectra, particularly for pMC5, were observed. Some of these changes involved residues widely separated in the primary structure. For instance, interactions involving Tyr-22 were influenced by the C-terminal amino acids suggesting that the N- and C-termini of mIL-6 are in close proximity. Equilibrium unfolding experiments indicated that pMC5 was 0.8 kcal/mol less stable than mIL-6, whereas pMC5H was 1.4 kcal/mol more stable. These studies emphasize the structural importance of the C-terminal amino acids of IL-6 and suggest that truncation or mutation of this region could lead to small but significant alterations in other regions of the molecule.
Publisher: Elsevier BV
Date: 08-2005
Publisher: Elsevier BV
Date: 09-1996
DOI: 10.1016/S0092-8674(00)80151-8
Abstract: Link modules are hyaluronan-binding domains found in proteins involved in the assembly of extracellular matrix, cell adhesion, and migration. The solution structure of the Link module from human TSG-6 was determined and found to consist of two alpha helices and two antiparallel beta sheets arranged around a large hydrophobic core. This defines the consensus fold for the Link module superfamily, which includes CD44, cartilage link protein, and aggrecan. The TSG-6 Link module was shown to interact with hyaluronan, and a putative binding surface was identified on the structure. A structural database search revealed close similarity between the Link module and the C-type lectin domain, with the predicted hyaluronan-binding site at an analogous position to the carbohydrate-binding pocket in E-selectin.
Publisher: Elsevier BV
Date: 02-1998
Abstract: The refolding kinetics of the chemically denatured SH3 domain of phosphatidylinositol 3'-kinase (PI3-SH3) have been monitored by real-time one-dimensional 1H NMR coupled with a variety of other biophysical techniques. These experiments indicate that the refolding kinetics of PI3-SH3 are biphasic. The slow phase (27 (+/- 8)% litude) is due to a population of substantially unfolded molecules with an incorrectly configured cis proline residue. The fast phase (73 (+/- 8)% litude) corresponds to the folding of protein molecules with proline residues in a trans configuration in the unfolded state. NMR experiments indicate that the first species populated after the initiation of folding exhibit poor chemical shift dispersion and have spectra very similar to that of the denatured protein in 8 M guanidine hydrochloride. Linear combinations of the first spectrum and of the spectrum of the native protein accurately reconstruct all of the spectra acquired during refolding. Consistent with this, native side-chain and backbone H alpha atom packing (NMR), secondary structure (far-UV circular dichroism), burial of aromatic residues (near-UV circular dichroism), intrinsic fluorescence and peptide binding activity are all recovered with effectively identical kinetics. Equilibrium unfolding and folding/unfolding kinetics yield, within experimental error, identical values for the free energy of unfolding (delta Gu-H2O = 3.38 kcal mol-1) and for the slope of the free energy of unfolding versus denaturant concentration (meq = 2.33 kcal mol-1 M-1). Together, these data provide strong evidence that PI3-SH3 folds without significant population of kinetic well-structured intermediates. That PI3-SH3 folds slowly (time constant 2.8 seconds in H2O at 20 degrees C) indicates that slow refolding is not always a consequence of kinetic traps but may be observed even when a protein appears to fold via a simple, two-state mechanism.
Publisher: Elsevier BV
Date: 06-2008
DOI: 10.1016/J.JMB.2008.04.066
Abstract: The commonly used anti-cancer drug chlorambucil is the primary treatment for patients with chronic lymphocytic leukaemia. Chlorambucil has been shown to be detoxified by human glutathione transferase Pi (GST P1-1), an enzyme that is often found over-expressed in cancer tissues. The allelic variants of GST P1-1 are associated with differing susceptibilities to leukaemia and differ markedly in their efficiency in catalysing glutathione (GSH) conjugation reactions. Here, we perform detailed kinetic studies of the allelic variants with the aid of three representative co-substrates. We show that the differing catalytic properties of the variants are highly substrate-dependent. We show also that all variants exhibit the same temperature stability in the range 10 degrees C to 45 degrees C. We have determined the crystal structures of GST P1-1 in complex with chlorambucil and its GSH conjugate for two of these allelic variants that have different residues at positions 104 and 113. Chlorambucil is found to bind in a non-productive mode to the substrate-binding site (H-site) in the absence of GSH. This result suggests that under certain stress conditions where GSH levels are low, GST P1-1 can inactivate the drug by sequestering it from the surrounding medium. However, in the presence of GSH, chlorambucil binds in the H-site in a productive mode and undergoes a conjugation reaction with GSH present in the crystal. The crystal structure of the GSH-chlorambucil complex bound to the *C variant is identical with the *A variant ruling out the hypothesis that primary structure differences between the variants cause structural changes at the active site. Finally, we show that chlorambucil is a very poor inhibitor of the enzyme in contrast to ethacrynic acid, which binds to the enzyme in a similar fashion but can act as both substrate and inhibitor.
Publisher: American Chemical Society (ACS)
Date: 22-05-2002
DOI: 10.1021/BI0121813
Abstract: The carboxyl terminus of the type-1 angiotensin II receptor (AT(1A)) is a focal point for receptor activation and deactivation. Synthetic peptides corresponding to the membrane-proximal, first 20 amino acids of the carboxyl terminus adopt an alpha-helical conformation in organic solvents, suggesting that the secondary structure of this region may be sensitive to hydrophobic environments. Using surface plasmon resonance, immobilized lipid chromatography, and circular dichroism, we examined whether this positively charged, hipathic alpha-helical region of the AT(1A) receptor can interact with lipid components in the cell membrane and thereby modulate local receptor attachment and structure. A synthetic peptide corresponding to the proximal region of the AT(1A) receptor carboxyl terminus (Leu(305) to Lys(325)) was shown by surface plasmon resonance to bind with high affinity to the negatively charged lipid, dimyristoyl L-alpha-phosphatidyl-DL-glycerol (DMPG), but poorly to the zwitterionic lipid, dimyristoyl L-alpha-phosphatidylcholine (DMPC). In contrast, a peptide analogue possessing substitutions at four lysine residues (corresponding to Lys(307,308,310,311)) displayed poor association with either lipid, indicating a crucial anionic component to the interaction. Circular dichroism analysis revealed that both the wild-type and substituted peptides possessed alpha-helical propensity in methanol and trifluoroethanol, while the wild-type peptide also adopted partially inserted helical structure in DMPG and DMPC liposomes. In contrast, the substituted peptide exhibited spectra that suggested the presence of beta-sheet and alpha-helical structure in both liposomes. Immobilized lipid chromatography was used to characterize the hydrophobic component of the membrane interaction, and the results demonstrated that hydrophobic and electrostatic interactions mediated the binding of the wild-type peptide but that the substituted peptide bound to the model membranes mainly via hydrophobic forces. We propose that, in intact AT(1A) receptors, the proximal carboxyl terminus associates with the cytoplasmic face of the cell membrane via a high-affinity, anionic phospholipid-specific tethering that serves to increase the hipathic helicity of this region. Such associations may be important for receptor function and common for G protein-coupled receptors.
Publisher: eLife Sciences Publications, Ltd
Date: 07-05-2015
DOI: 10.7554/ELIFE.05142
Abstract: Antifreeze proteins (AFPs) protect certain cold-adapted organisms from freezing to death by selectively adsorbing to internal ice crystals and inhibiting ice propagation. The molecular details of AFP adsorption-inhibition is uncertain but is proposed to involve the Gibbs–Thomson effect. Here we show by using unbiased molecular dynamics simulations a protein structure-function mechanism for the spruce budworm Choristoneura fumiferana AFP, including stereo-specific binding and consequential melting and freezing inhibition. The protein binds indirectly to the prism ice face through a linear array of ordered water molecules that are structurally distinct from the ice. Mutation of the ice binding surface disrupts water-ordering and abolishes activity. The adsorption is virtually irreversible, and we confirm the ice growth inhibition is consistent with the Gibbs–Thomson law.
Publisher: Microbiology Society
Date: 08-2005
Abstract: Human cytomegalovirus (HCMV) resistance to antivirals is a significant clinical problem. Murine cytomegalovirus (MCMV) infection of mice is a well-described animal model for in vivo studies of CMV pathogenesis, although the mechanisms of MCMV antiviral susceptibility need elucidation. Mutants resistant to nucleoside analogues aciclovir, adefovir, cidofovir, ganciclovir, penciclovir and valaciclovir, and the pyrophosphate analogue foscarnet were generated by in vitro passage of MCMV (Smith) in increasing concentrations of antiviral. All MCMV antiviral resistant mutants contained DNA polymerase mutations identical or similar to HCMV DNA polymerase mutations known to confer antiviral resistance. Mapping of the mutations onto an MCMV DNA polymerase three-dimensional model generated using the Thermococcus gorgonarius Tgo polymerase crystal structure showed that the DNA polymerase mutations potentially confer resistance through changes in regions surrounding a catalytic aspartate triad. The ganciclovir-, penciclovir- and valaciclovir-resistant isolates also contained mutations within MCMV M97 identical or similar to recognized GCV-resistant mutations of HCMV UL97 protein kinase, and demonstrated cross-resistance to antivirals of the same class. This strongly suggests that MCMV M97 has a similar role to HCMV UL97 in the phosphorylation of nucleoside analogue antivirals. All MCMV mutants demonstrated replication-impaired phenotypes, with the lowest titre and plaque size observed for isolates containing mutations in both DNA polymerase and M97. These findings indicate DNA polymerase and protein kinase regions of potential importance for antiviral susceptibility and replication. The similarities between MCMV and HCMV mutations that arise under antiviral selective pressure increase the utility of MCMV as a model for in vivo studies of CMV antiviral resistance.
Publisher: Elsevier BV
Date: 11-2008
Publisher: Wiley
Date: 19-08-2019
Abstract: Most structure-based drug discovery methods utilize crystal structures of receptor proteins. Crystal engineering, on the other hand, utilizes the wealth of chemical information inherent in small-molecule crystal structures in the Cambridge Structural Database (CSD). We show that the interaction surfaces and shapes of molecules in experimentally determined small-molecule crystal structures can serve as effective tools in drug discovery. Our description of the shape and interaction propensities of molecules in their crystal structures can be used to screen them for specific binding compatibility with protein targets, as demonstrated through the high-throughput profiling of around 138 000 small-molecule structures in the CSD and a series of drug-protein crystal structures. Electron-density-based intermolecular boundary surfaces in small-molecule crystal structures and in target-protein pockets are utilized to identify potential ligand molecules from the CSD based on 3D shape and intermolecular interaction matching.
Publisher: Elsevier BV
Date: 11-2014
DOI: 10.1016/J.IBMB.2014.08.008
Abstract: Extensive use of older generation insecticides may result in pre-existing cross-resistance to new chemical classes acting at the same target site. Phenylpyrazole insecticides block inhibitory neurotransmission in insects via their action on ligand-gated chloride channels (LGCCs). Phenylpyrazoles are broad-spectrum insecticides widely used in agriculture and domestic pest control. So far, all identified cases of target site resistance to phenylpyrazoles are based on mutations in the Rdl (Resistance to dieldrin) LGCC subunit, the major target site for cyclodiene insecticides. We examined the role that mutations in Rdl have on phenylpyrazole resistance in Drosophila melanogaster, exploring naturally occurring variation, and generating predicted resistance mutations by mutagenesis. Natural variation at the Rdl locus in inbred strains of D. melanogaster included gene duplication, and a line containing two Rdl mutations found in a highly resistant line of Drosophila simulans. These mutations had a moderate impact on survival following exposure to two phenylpyrazoles, fipronil and pyriprole. Homology modelling suggested that the Rdl chloride channel pore contains key residues for binding fipronil and pyriprole. Mutagenesis of these sites and assessment of resistance in vivo in transgenic lines showed that amino acid identity at the Ala(301) site influenced resistance levels, with glycine showing greater survival than serine replacement. We confirm that point mutations at the Rdl 301 site provide moderate resistance to phenylpyrazoles in D. melanogaster. We also emphasize the beneficial aspects of testing predicted mutations in a whole organism to validate a candidate gene approach.
Publisher: Wiley
Date: 10-1996
Abstract: The elucidation of a relationship between the thermodynamic parameters and the structural changes accompanying biomolecular interactions could lead to predictive algorithms. For ex le, based on some knowledge of the structure of a target molecule the affinities of ligands could be determined with obvious implications for the pharmaceutical industry. In attempting to relate the thermodynamic and structural changes on formation of a protein-DNA complex, the correlation between change in heat capacity and burial of surface area has proved successful. However, this correlation appears to break down when water molecules are included in the binding interface. Here we present data that support the hypothesis that bound water molecules have to be considered as contributing to the change in heat capacity and could, thus, be used in ligand design.
Publisher: Springer Science and Business Media LLC
Date: 13-03-2014
DOI: 10.1038/NRC3690
Abstract: Historically, targeting protein-protein interactions with small molecules was not thought possible because the corresponding interfaces were considered mostly flat and featureless and therefore 'undruggable'. Instead, such interactions were targeted with larger molecules, such as peptides and antibodies. However, the past decade has seen encouraging breakthroughs through the refinement of existing techniques and the development of new ones, together with the identification and exploitation of unexpected aspects of protein-protein interaction surfaces. In this Review, we describe some of the latest techniques to discover modulators of protein-protein interactions and how current drug discovery approaches have been adapted to successfully target these interfaces.
Publisher: Wiley
Date: 29-12-2018
DOI: 10.1002/ART.40369
Abstract: Endoplasmic reticulum aminopeptidase 1 ( ERAP ‐1) and ERAP ‐2 , encoded on chromosome 5q15, trim endogenous peptides for HLA ‐mediated presentation to the immune system. Polymorphisms in ERAP 1 and/or ERAP 2 are strongly associated with several immune‐mediated diseases with specific HLA backgrounds, implicating altered peptide handling and presentation as prerequisites for autoreactivity against an arthritogenic peptide. Given the thorough characterization of disease risk–associated polymorphisms that alter ERAP activity, this study aimed instead to interrogate the expression effect of chromosome 5q15 polymorphisms to determine their effect on ERAP isoform and protein expression. RNA sequencing and genotyping across chromosome 5q15 were performed to detect genetic variants in ERAP 1 and ERAP 2 associated with altered total gene and isoform‐specific expression. The functional implication of a putative messenger RNA splice‐altering variant on ERAP ‐1 protein levels was validated using mass spectrometry. Polymorphisms associated with ankylosing spondylitis ( AS ) significantly influenced the transcript and protein expression of ERAP‐1 and ERAP‐2. Disease risk–associated polymorphisms in and around both genes were also associated with increased gene expression. Furthermore, key risk‐associated ERAP 1 variants were associated with altered transcript splicing, leading to allele‐dependent alternate expression of 2 distinct isoforms and significant differences in the type of ERAP ‐1 protein produced. In accordance with studies demonstrating that polymorphisms that increase aminopeptidase activity predispose to immune disease, the increased risk also attributed to increased expression of ERAP 1 and ERAP 2 supports the notion of using aminopeptidase inhibition to treat AS and other ERAP ‐associated conditions.
Publisher: Elsevier BV
Date: 12-2011
DOI: 10.1016/J.BMCL.2011.09.109
Abstract: PDE4 inhibitors have been identified as therapeutic targets for a variety of conditions, particularly inflammatory diseases. We have serendipitously identified a novel class of phosphodiesterase 4 (PDE4) inhibitor during a study to discover antagonists of the parathyroid hormone receptor. X-ray crystallographic studies of PDE4D2 complexed to four potent inhibitors reveal the atomic details of how they inhibit the enzyme and a notable contrast to another recently reported thiophene-based inhibitor.
Publisher: Wiley
Date: 1994
DOI: 10.1111/J.1432-1033.1994.TB19919.X
Abstract: Two synthetic peptides corresponding to the C‐terminal 19 residues of human and murine interleukin‐6, respectively, have been synthesized and their structures in solution investigated using high‐resolution 1 H‐NMR spectroscopy. Both peptides show a marked dependence of chemical‐shift dispersion on pH, with a greater degree of structure apparent above pH 4.5, where their glutamate carboxyl groups are ionised. In purely aqueous solution, neither peptide adopts a well‐defined structure, although the murine peptide has characteristics of a nascent helix. Titration of the murine peptide with trifluoroethanol produced a significant increase in structure, which was then investigated using two‐dimensional NMR. In 50% (by vol.) trifluoroethanol the murine peptide consists of a well‐defined central helix of 12 residues with unstructured N‐terminal and C‐terminal regions. These observations lend experimental support to the current model of the interleukin‐6 structure, which proposes a four‐helical bundle with the last helix encompassing the C‐terminal 20–30 residues. Furthermore, the fact that synthetic peptides corresponding to part of the putative receptor‐binding surface of interleukin‐6 are able to adopt a similar conformation in solution to that proposed for the intact protein suggests that such peptide analogues should be useful starting points in the design of peptide agonists and antagonists of interleukin‐6.
Publisher: American Chemical Society (ACS)
Date: 16-02-2012
DOI: 10.1021/ML2002955
Publisher: Springer Science and Business Media LLC
Date: 23-01-2017
DOI: 10.1038/NCOMMS14188
Abstract: C-reactive protein (CRP) concentrations rise in response to tissue injury or infection. Circulating pentameric CRP (pCRP) localizes to damaged tissue where it leads to complement activation and further tissue damage. In-depth knowledge of the pCRP activation mechanism is essential to develop therapeutic strategies to minimize tissue injury. Here we demonstrate that pCRP by binding to cell-derived microvesicles undergoes a structural change without disrupting the pentameric symmetry (pCRP*). pCRP* constitutes the major CRP species in human-inflamed tissue and allows binding of complement factor 1q (C1q) and activation of the classical complement pathway. pCRP*–microvesicle complexes lead to enhanced recruitment of leukocytes to inflamed tissue. A small-molecule inhibitor of pCRP (1,6-bis(phosphocholine)-hexane), which blocks the pCRP–microvesicle interactions, abrogates these proinflammatory effects. Reducing inflammation-mediated tissue injury by therapeutic inhibition might improve the outcome of myocardial infarction, stroke and other inflammatory conditions.
Publisher: Springer Science and Business Media LLC
Date: 23-04-2014
DOI: 10.1038/SREP04765
Publisher: Elsevier BV
Date: 08-2021
Publisher: Springer Science and Business Media LLC
Date: 26-01-2018
DOI: 10.1038/S41467-017-02633-7
Abstract: The interleukin-3 (IL-3) receptor is a cell-surface heterodimer that links the haemopoietic, vascular and immune systems and is overexpressed in acute and chronic myeloid leukaemia progenitor cells. It belongs to the type I cytokine receptor family in which the α-subunits consist of two fibronectin III-like domains that bind cytokine, and a third, evolutionarily unrelated and topologically conserved, N-terminal domain (NTD) with unknown function. Here we show by crystallography that, while the NTD of IL3Rα is highly mobile in the presence of IL-3, it becomes surprisingly rigid in the presence of IL-3 K116W. Mutagenesis, biochemical and functional studies show that the NTD of IL3Rα regulates IL-3 binding and signalling and reveal an unexpected role in preventing spontaneous receptor dimerisation. Our work identifies a dual role for the NTD in this cytokine receptor family, protecting against inappropriate signalling and dynamically regulating cytokine receptor binding and function.
Publisher: Elsevier BV
Date: 02-2015
DOI: 10.1016/J.BMCL.2014.11.018
Abstract: Respiratory syncytial virus (RSV) is a major cause of respiratory tract infections in infants, young children and adults. Compound 1a (9b-(4-chlorophenyl)-1-(4-fluorobenzoyl)-1,2,3,9b-tetrahydro-5H-imidazo[2,1-a]isoindol-5-one) was identified as an inhibitor of A and B strains of RSV targeting the fusion glycoprotein. SAR was developed by systematic exploration of the phenyl (R(1)) and benzoyl (R(2)) groups. Furthermore, introduction of a nitrogen at the 8-position of the tricyclic core resulted in active analogues with improved properties (aqueous solubility, protein binding and logD) and excellent rat pharmacokinetics (e.g., rat oral bioavailability of 89% for compound 17).
Publisher: No publisher found
Date: 2017
DOI: 10.1002/PRO.3084
Publisher: Elsevier BV
Date: 11-2001
Publisher: Elsevier BV
Date: 2000
Publisher: eLife Sciences Publications, Ltd
Date: 27-06-2022
Publisher: American Chemical Society (ACS)
Date: 02-1998
DOI: 10.1021/BI972075U
Publisher: Wiley
Date: 06-1990
DOI: 10.1111/J.1432-1033.1990.TB15579.X
Abstract: The complete amino acid sequence of the cardiac stimulatory and haemolytic protein tenebrosin-C, from the Australian sea anemone Actinia tenebrosa, has been determined by Edman degradation of the intact molecule and fragments produced by treatment of the polypeptide chain with cyanogen bromide and enzymatic cleavage with endoproteinase Asp-N, thermolysin and trypsin. The molecule is a single-chain polypeptide consisting of 179 amino acid residues with a calculated molecular mass of 19,797 Da. Tenebrosin-C shows a high degree of amino acid sequence similarity (63%) with Stoichactis helianthus cytolysin III [Blumenthal, K. M. and Kem, W. R. (1983) J. Biol. Chem. 258, 5574-5581] and is identical to a partial sequence (90 residues) reported for equinatoxin, a cardiostimulatory and haemolytic protein isolated from the European sea anemone Actinia equina [Ferlan, I. and Jackson, K. (1983) Toxicon Suppl. 3, 141-144]. No amino acid sequence similarity was detected between tenebrosin-C and other protein sequences stored in available databases. The predicted secondary structure of tenebrosin-C suggests that it is a compact, highly structured molecule.
Publisher: Proceedings of the National Academy of Sciences
Date: 21-09-2021
Abstract: Here, we describe inhibitors of the Plasmodium proteasome, an enzymatic complex that malaria parasites rely on to degrade proteins. Starting from inhibitors developed to treat cancer, derivatives were designed and synthesized with the aim of increasing potency against the Plasmodium proteasome and decreasing activity against the human enzyme. Biochemical and cellular assays identified compounds that exhibit selectivity and potency, both in vitro and in vivo, at different stages of the parasite’s lifecycle. Cryo-electron microscopy revealed that the inhibitors bind in a hydrophobic pocket that is structurally different in the human proteasome—underpinning their selectivity. The work will help develop antimalarial therapeutics, which are desperately needed to treat a disease that kills nearly half a million people annually.
Publisher: Elsevier BV
Date: 09-2014
DOI: 10.1016/J.BMCL.2014.07.037
Abstract: A series of dual-targeting, alcohol-containing benzothiazoles has been identified with superior antibacterial activity and drug-like properties. Early lead benzothiazoles containing carboxylic acid moieties showed efficacy in a well-established in vivo model, but inferior drug-like properties demanded modifications of functionality capable of demonstrating superior efficacy. Eliminating the acid group in favor of hydrophilic alcohol moieties at C(5), as well as incorporating solubilizing groups at the C(7) position of the core ring provided potent, broad-spectrum Gram-positive antibacterial activity, lower protein binding, and markedly improved efficacy in vivo.
Publisher: Wiley
Date: 16-06-2011
Abstract: Platinum-based cancer drugs, such as cisplatin, are highly effective chemotherapeutic agents used extensively for the treatment of solid tumors. However, their effectiveness is limited by drug resistance, which, in some cancers, has been associated with an overexpression of pi class glutathione S-transferase (GST P1-1), an important enzyme in the mercapturic acid detoxification pathway. Ethacraplatin (EA-CPT), a trans-Pt(IV) carboxylate complex containing ethacrynate ligands, was designed as a platinum cancer metallodrug that could also target cytosolic GST enzymes. We previously reported that EA-CPT was an excellent inhibitor of GST activity in live mammalian cells compared to either cisplatin or ethacrynic acid. In order to understand the nature of the drug-protein interactions between EA-CPT and GST P1-1, and to obtain mechanistic insights at a molecular level, structural and biochemical investigations were carried out, supported by molecular modeling analysis using quantum mechanical/molecular mechanical methods. The results suggest that EA-CPT preferentially docks at the dimer interface at GST P1-1 and subsequent interaction with the enzyme resulted in docking of the ethacrynate ligands at both active sites (in the H-sites), with the Pt moiety remaining bound at the dimer interface. The activation of the inhibitor by its target enzyme and covalent binding accounts for the strong and irreversible inhibition of enzymatic activity by the platinum complex.
Publisher: Elsevier BV
Date: 08-2003
Publisher: Elsevier BV
Date: 06-1995
DOI: 10.1016/0167-4838(95)00023-N
Abstract: The effects of solvent, pH and temperature on the 1H-NMR spectra of recombinant murine interleukin-6 (IL-6) are described. Assignments made from two-dimensional homonuclear spectra are presented for resonances of the fifteen aromatic amino-acid side chains. A time-dependent loss of intensity was observed for all resonances in the spectrum of IL-6, probably as a result of aggregation. This aggregation is markedly temperature-dependent. The pKa values of the four histidine residues in murine IL-6 has been measured one has a value of 5.5, approx. one pH unit less than the value exhibited by the other three. Analysis of the NOESY spectra has allowed a preliminary characterisation of the nature of interactions among the aromatic side chains within the protein fold. 1H and 15N resonances of residues Thr-4 to Val-21 are assigned from three-dimensional 1H-15N correlated spectroscopy, and evidence is presented for these residues comprising a mobile N-terminal tail with little ordered structure. An N-terminal mutant lacking the first 22 residues of the murine IL-6 sequence and known to possess full biological activity was also examined and shown to have essentially retained the tertiary fold of the native molecule.
Publisher: Wiley
Date: 1999
DOI: 10.1110/PS.8.10.1962
Abstract: We have adopted nanoflow electrospray ionization mass spectrometry (ESI-MS) and isothermal titration calorimetry (ITC) to probe the mechanism of peptide recognition by the SH2 domain from the Src family tyrosine kinase protein, Fyn. This domain is involved in the mediation of intracellular signal transduction pathways by interaction with proteins containing phosphorylated tyrosine (Y*) residues. The binding of tyrosyl phosphopeptides can mimic these interactions. Specificity in these interactions has been attributed to the interaction of the Y* and residues proximal and C-terminal to it. Previous studies have established that for specific binding with Fyn, the recognition sequence consists of pTyr-Glu-Glu-Ile. The specific interactions involve the binding of Y* with the ionic, and the Y* + 3 Ile residue with the hydrophobic binding pockets on the surface of the Fyn SH2 domain. In this work, a variation in the Y* + 3 residue of this high-affinity sequence was observed to result in changes in the relative binding affinities as determined in solution (ITC) and in the gas phase (nanoflow ESI-MS). X-ray analysis shows that a feature of the Src family SH2 domains is the involvement of water molecules in the peptide binding site. Under the nanoflow ESI conditions, water molecules appear to be maintained in the Fyn SH2-ligand complex. Compelling evidence for these molecules being incorporated in the SH2-peptide interface is provided by the prevalence of the peaks assigned to water-bound over the water-free complex at high-energy conditions. Thus, the stability of water protein-ligand complex appears to be intimately linked to the presence of water.
Publisher: Elsevier BV
Date: 06-1996
DOI: 10.1016/S0969-2126(96)00076-7
Abstract: The Src family of tyrosine kinases is involved in the propagation of intracellular signals from many transmembrane receptors. Each member of the family contains two domains that regulate interactions with other molecules, one of which is the Src homology 3 (SH3) domain. Although structures have previously been determined for SH3 domains, and ideas about peptide-binding modes have been proposed, their physiological role is still unclear. We have determined the solution structure of the SH3 domain from the Src family tyrosine kinase Fyn in two forms: unbound and complexed with a peptide corresponding to a putative ligand sequence from phosphatidylinositol 3' kinase. Fyn SH3 shows the typical SH3 topology of two perpendicular three-stranded beta sheets and a single turn of 3(10) helix. The interaction of SH3 with three potential ligand peptides was investigated, demonstrating that they all bind to the same site on the molecule. A previous model for ligand binding to SH3 domains predicts binding in one of two orientations (class I or II), each characterized by a consensus sequence. The ligand with the closest match to the class I consensus sequence bound with highest affinity and in the predicted orientation. The Fyn SH3 domain has a well-defined structure in solution. The relative binding affinities of the three ligand peptides and their orientation within the Fyn SH3 complex were consistent with recently proposed models for the binding of 'consensus' polyproline sequences. Although the affinities of consensus and non-consensus peptides are different, the degree of difference is not very large, suggesting that SH3 domains bind to polyproline peptides in a promiscuous manner.
Publisher: Elsevier BV
Date: 10-1997
DOI: 10.1016/S0969-2126(97)00283-9
Abstract: SH2 domains are found in a variety of signal transduction proteins they bind phosphotyrosine-containing sequences, allowing them to both recognize target molecules and regulate intramolecular kinase activity. Fyn is a member of the Src family of tyrosine kinases that are involved in signal transduction by association with a number of membrane receptors. The kinase activity of these signalling proteins is modulated by switching the binding mode of their SH2 and SH3 domains from intramolecular to intermolecular. The molecular basis of the signalling roles observed for different Src family members is still not well understood although structures have been determined for the SH2 domains of other Src family molecules, this is the first structure of the Fyn SH2 domain. The structure of the Fyn SH2 domain in complex with a phosphotyrosyl peptide (EPQpYEEIPIYL) was determined by high resolution NMR spectroscopy. The overall structure of the complex is analogous to that of other SH2-peptide complexes. Noteworthy aspects of the structure are: the BG loop, which contacts the bound peptide, contains a type-I' turn a capping-box-like interaction is present at the N-terminal end of helix alpha A cis-trans isomerization of the Val beta G1-Pro beta G2 peptide bond causes conformational heterogeneity of residues near the N and C termini of the domain. Comparison of the Fyn SH2 domain structure with other structures of SH2 domains highlights several interesting features. Conservation of helix capping interactions among various SH2 domains is suggestive of a role in protein stabilisation. The presence of a type-I' turn in the BG loop, which is dependent on the presence of a glycine residue at position BG3, is indicative of a binding pocket, characteristic of the Src family, SykC and Abl, rather than a binding groove found in PLC-gamma 1C, p85 alpha N and Shc, for ex le.
Publisher: American Society for Microbiology
Date: 15-07-2003
DOI: 10.1128/JB.185.14.4057-4065.2003
Abstract: Bioinformatic analyses of whole genome sequences highlight the problem of identifying the biochemical and cellular functions of many gene products that are at present uncharacterized. The open reading frame Rv3853 from Mycobacterium tuberculosis has been annotated as menG and assumed to encode an S -adenosylmethionine (SAM)-dependent methyltransferase that catalyzes the final step in menaquinone biosynthesis. The Rv3853 gene product has been expressed, refolded, purified, and crystallized in the context of a structural genomics program. Its crystal structure has been determined by isomorphous replacement and refined at 1.9 Å resolution to an R factor of 19.0% and R free of 22.0%. The structure strongly suggests that this protein is not a SAM-dependent methyltransferase and that the gene has been misannotated in this and other genomes that contain homologs. The protein forms a tightly associated, disk-like trimer. The monomer fold is unlike that of any known SAM-dependent methyltransferase, most closely resembling the phosphohistidine domains of several phosphotransfer systems. Attempts to bind cofactor and substrate molecules have been unsuccessful, but two adventitiously bound small-molecule ligands, modeled as tartrate and glyoxalate, are present on each monomer. These may point to biologically relevant binding sites but do not suggest a function. In silico screening indicates a range of ligands that could occupy these and other sites. The nature of these ligands, coupled with the location of binding sites on the trimer, suggests that proteins of the Rv3853 family, which are distributed throughout microbial and plant species, may be part of a larger assembly binding to nucleic acids or proteins.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 16-05-2014
Abstract: Signaling from JAK (Janus kinase) protein kinases to STAT (signal transducers and activators of transcription) transcription factors is key to many aspects of biology and medicine, yet the mechanism by which cytokine receptors initiate signaling is enigmatic. We present a complete mechanistic model for activation of receptor-bound JAK2, based on an archetypal cytokine receptor, the growth hormone receptor. For this, we used fluorescence resonance energy transfer to monitor positioning of the JAK2 binding motif in the receptor dimer, substitution of the receptor extracellular domains with Jun zippers to control the position of its transmembrane (TM) helices, atomistic modeling of TM helix movements, and docking of the crystal structures of the JAK2 kinase and its inhibitory pseudokinase domain with an opposing kinase-pseudokinase domain pair. Activation of the receptor dimer induced a separation of its JAK2 binding motifs, driven by a ligand-induced transition from a parallel TM helix pair to a left-handed crossover arrangement. This separation leads to removal of the pseudokinase domain from the kinase domain of the partner JAK2 and pairing of the two kinase domains, facilitating trans-activation. This model may well generalize to other class I cytokine receptors.
Publisher: Proceedings of the National Academy of Sciences
Date: 20-06-2019
Abstract: Cisplatin [ cis- diamminedichloroplatinum(II) ( cis -DDP)] is one of the most successful anticancer agents effective against a wide range of solid tumors. However, its use is restricted by side effects and/or by intrinsic or acquired drug resistance. Here, we probed the role of glutathione transferase (GST) P1-1, an antiapoptotic protein often overexpressed in drug-resistant tumors, as a cis -DDP–binding protein. Our results show that cis -DDP is not a substrate for the glutathione (GSH) transferase activity of GST P1-1. Instead, GST P1-1 sequesters and inactivates cisplatin with the aid of 2 solvent-accessible cysteines, resulting in protein subunits cross-linking, while maintaining its GSH-conjugation activity. Furthermore, it is well known that GST P1-1 binding to the c-Jun N-terminal kinase (JNK) inhibits JNK phosphorylation, which is required for downstream apoptosis signaling. Thus, in turn, GST P1-1 overexpression and Pt-induced subunit cross-linking could modulate JNK apoptotic signaling, further confirming the role of GST P1-1 as an antiapoptotic protein.
Publisher: Wiley
Date: 26-04-2006
Abstract: LIM kinase 1 (LIMK1) is a serine protein kinase that regulates the actin cytoskeleton by phosphorylation and inactivation of actin depolymerizing factor cofilin. LIMK1 activity is regulated by the Rho-GTPases via their serine/threonine kinase effectors Rho-kinase and p21-activated kinases 1 and 4 that phosphorylate LIMK1 on threonine 508 in its activation loop. The purpose of this study was to elucidate the pathway leading to the stability of LIMK1, a protein with a half-life of approximately 20 h. Because the half-life of kinase-dead LIMK1 is only 4 h, it is suggestive that trans- or auto-phosphorylation is responsible for the stabilization of LIMK1. Using known Hsp90 inhibitors, we have shown that the half-life of LIMK1 in cells depends on the presence of active Hsp90. Furthermore, endogenous LIMK1 coimmunoprecipitated with endogenous Hsp90 and this interaction promoted LIMK1 homodimer formation as seen by cross-linking experiments. Hsp90 binds LIMK1 via a recognition sequence within the LIMK1 kinase domain, homologous to that of ErbB-2. Mutation of a proline residue within this sequence to glutamic acid reduces its interaction with Hsp90, inhibits homodimer formation, and reduces its half-life to 4 h. These findings implicate Hsp90 in the stabilization of LIMK1 by promoting homodimer formation and transphosphorylation.
Publisher: Springer Science and Business Media LLC
Date: 18-11-2022
Publisher: Elsevier BV
Date: 06-2020
Publisher: Elsevier BV
Date: 11-2014
DOI: 10.1016/J.BMCL.2014.09.030
Abstract: Previous investigations identified 2'-C-Me-branched ribo-C-nucleoside adenosine analogues, 1, which contains a pyrrolo[2,1-f][1,2,4]triazin-4-amine heterocyclic base, and 2, which contains an imidazo[2,1-f][1,2,4]triazin-4-amine heterocyclic base as two compounds with promising anti-HCV in vitro activity. This Letter describes the synthesis and evaluation of a series of novel analogues of these compounds substituted at the 2-, 7-, and 8-positions of the heterocyclic bases. A number of active new HCV inhibitors were identified but most compounds also demonstrated unacceptable cytotoxicity. However, the 7-fluoro analogue of 1 displayed good potency with a promising cytotherapeutic margin.
Publisher: Springer Science and Business Media LLC
Date: 16-08-2018
Publisher: Springer Science and Business Media LLC
Date: 08-2002
DOI: 10.1007/S00249-002-0219-1
Abstract: Many plasma membrane chloride channels have been cloned and characterized in great detail. In contrast, very little is known about intracellular chloride channels. Members of a novel class of such channels, called the CLICs (chloride intracellular channels), have been identified over the last few years. A striking feature of the CLIC family of ion channels is that they can exist in a water-soluble state as well as a membrane-bound state. A major step forward in understanding the functioning of these channels has been the recent crystal structure determination of one family member, CLIC1. The structure confirms that CLICs are members of the glutathione S-transferase superfamily and provides clues as to how CLICs can insert into membranes to form chloride channels.
Publisher: MDPI AG
Date: 24-11-2022
DOI: 10.3390/V14122620
Abstract: SARS-CoV-2 is the causative agent of the COVID-19 pandemic. Vaccination, supported by social and public health measures, has proven efficacious for reducing disease severity and virus spread. However, the emergence of highly transmissible viral variants that escape prior immunity highlights the need for additional mitigation approaches. Heparin binds the SARS-CoV-2 spike protein and can inhibit virus entry and replication in susceptible human cell lines and bronchial epithelial cells. Primary infection predominantly occurs via the nasal epithelium, but the nasal cell biology of SARS-CoV-2 is not well studied. We hypothesized that prophylactic intranasal administration of heparin may provide strain-agnostic protection for household contacts or those in high-risk settings against SARS-CoV-2 infection. Therefore, we investigated the ability of heparin to inhibit SARS-CoV-2 infection and replication in differentiated human nasal epithelial cells and showed that prolonged exposure to heparin inhibits virus infection. Furthermore, we establish a method for PCR detection of SARS-CoV-2 viral genomes in heparin-treated s les that can be adapted for the detection of viruses in clinical studies.
Publisher: EMBO
Date: 05-12-2022
Publisher: Elsevier BV
Date: 06-2002
DOI: 10.1016/S0968-0004(02)02092-3
Abstract: The GABA(A) receptor is the primary mediator of inhibitory neurotransmission in the brain and is a major target for neuromodulatory drugs such as benzodiazepines, barbiturates, ethanol and anaesthetics. However, our understanding of the molecular details of this receptor has been limited by a lack of high-resolution structural information. This article presents a new model for the extracellular, ligand-binding domain of the GABA(A) receptor, that is based on the recently determined structure of a soluble acetylcholine-binding protein. The model puts existing mutational and biochemical data into a three-dimensional context, shows details of the GABA- and benzodiazepine-binding sites, and highlights the importance of other regions in allosteric conformational change. This provides a new perspective on existing data and an exciting new framework for understanding this important family of receptors.
Publisher: Portland Press Ltd.
Date: 21-09-2018
DOI: 10.1042/BST20180202
Abstract: The first protein structures revealed a complex web of weak interactions stabilising the three-dimensional shape of the molecule. Small molecule ligands were then found to exploit these same weak binding events to modulate protein function or act as substrates in enzymatic reactions. As the understanding of ligand–protein binding grew, it became possible to firstly predict how and where a particular small molecule might interact with a protein, and then to identify putative ligands for a specific protein site. Computer-aided drug discovery, based on the structure of target proteins, is now a well-established technique that has produced several marketed drugs. We present here an overview of the various methodologies being used for structure-based computer-aided drug discovery and comment on possible future developments in the field.
Publisher: CSIRO Publishing
Date: 2013
DOI: 10.1071/CH13280
Abstract: The design of a suitable library is an essential prerequisite to establish a fragment-based screening capability. Several pharmaceutical companies have described their approaches to establishing fragment libraries however there are few detailed reports of both design and analysis of performance for a fragment library maintained in an academic setting. Here we report our efforts towards the design of a fragment library for nuclear magnetic resonance spectroscopy-based screening, demonstrate the performance of the library through analysis of 14 screens, and present a comparison to previously reported fragment libraries.
Publisher: CSIRO Publishing
Date: 2014
DOI: 10.1071/CH14397
Abstract: Phosphodiesterase 4 (PDE4), the primary cyclic AMP-hydrolysing enzyme in cells, is a promising drug target for a wide range of mental disorders including Alzheimer's and Huntington's diseases, schizophrenia, and depression, plus a range of inflammatory diseases including chronic obstructive pulmonary disease, asthma, and rheumatoid arthritis. However, targeting PDE4 is complicated by the fact that the enzyme is encoded by four very closely related genes, together with 20 distinct isoforms as a result of mRNA splicing, and inhibition of some of these isoforms leads to intolerable side effects in clinical trials. With almost identical active sites between the isoforms, X-ray crystallography has played a critical role in the discovery and development of safer PDE4 inhibitors. Here we describe our discovery of a novel class of highly potent PDE4 via a ‘virtuous’ cycle of structure-based drug design and serendipity.
Publisher: Springer Science and Business Media LLC
Date: 08-2002
DOI: 10.1007/S00249-002-0229-Z
Abstract: Melittin is a cytolytic peptide whose biological activity is lost upon binding to a six-residue peptide, Ac-IVIFDC-NH(2), with which it forms a highly insoluble complex. As a result, the structural analysis of the interaction between the two peptides is difficult. Solid-state NMR spectroscopy was used to study the interaction between melittin and the peptide inhibitor. Location of the binding site in the melittin-inhibitor complex was determined using lanthanide ions, which quench NMR resonances from molecular sites that are in close proximity to the unique ion binding site. Our results indicated that the inhibitor binding site in melittin is near Leu13, Leu16 and Ile17, but not near Leu6 or Val8. On the basis of these data we propose that the inhibitor binds to melittin in the vicinity of Ala15 to Trp19 and prevents insertion of melittin into cell membranes by disrupting the helical structure. Supporting evidence for this model was produced by determining the distance, using rotational resonance NMR, between the [1-(13)C] of Leu13 in melittin and the [3-(13)C] of Phe4 in the inhibitor.
Publisher: Elsevier BV
Date: 09-2005
Publisher: Springer Science and Business Media LLC
Date: 25-09-2015
DOI: 10.1038/SREP14352
Abstract: 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.
Publisher: Wiley
Date: 26-12-2015
DOI: 10.1002/PRO.2604
Publisher: Wiley
Date: 14-07-2022
DOI: 10.1002/IUB.2661
Abstract: The cholesterol‐dependent cytolysins (CDCs) are a major family of bacterial pore‐forming proteins secreted as virulence factors by Gram‐positive bacterial species. CDCs are produced as soluble, monomeric proteins that bind specifically to cholesterol‐rich membranes, where they oligomerize into ring‐shaped pores of more than 30 monomers. Understanding the details of the steps the toxin undergoes in converting from monomer to a membrane‐spanning pore is a continuing challenge. In this review we summarize what we know about CDCs and highlight the remaining outstanding questions that require answers to obtain a complete picture of how these toxins kill cells.
Publisher: Proceedings of the National Academy of Sciences
Date: 02-02-2015
Abstract: Bacterial pathogens produce pore-forming toxins that damage eukaryotic membranes, whereas the pore-forming immune defense proteins produced by vertebrates can damage bacterial membranes. Despite the opposite functions of these proteins in pathogenesis or protection, many use a common pore-forming mechanism whereby membrane-bound monomers oligomerize into a circular structure, termed the prepore, which then assembles a β-barrel structure that punches a hole in the membrane. Here we show that once the prepore is assembled, an intermolecular electrostatic interaction is established that drives the formation of the pore. This mechanism is likely to be used by toxins and other pore-forming proteins that span the biological domains of life.
Publisher: Frontiers Media SA
Date: 13-09-2022
DOI: 10.3389/FIMMU.2022.1002652
Abstract: C-reactive protein (CRP) is a member of the highly conserved pentraxin superfamily of proteins and is often used in clinical practice as a marker of infection and inflammation. There is now increasing evidence that CRP is not only a marker of inflammation, but also that destabilized isoforms of CRP possess pro-inflammatory and pro-thrombotic properties. CRP circulates as a functionally inert pentameric form (pCRP), which relaxes its conformation to pCRP* after binding to phosphocholine-enriched membranes and then dissociates to monomeric CRP (mCRP). with the latter two being destabilized isoforms possessing highly pro-inflammatory features. pCRP* and mCRP have significant biological effects in regulating many of the aspects central to pathogenesis of atherothrombosis and venous thromboembolism (VTE), by directly activating platelets and triggering the classical complement pathway. Importantly, it is now well appreciated that VTE is a consequence of thromboinflammation. Accordingly, acute VTE is known to be associated with classical inflammatory responses and elevations of CRP, and indeed VTE risk is elevated in conditions associated with inflammation, such as inflammatory bowel disease, COVID-19 and sepsis. Although the clinical data regarding the utility of CRP as a biomarker in predicting VTE remains modest, and in some cases conflicting, the clinical utility of CRP appears to be improved in subsets of the population such as in predicting VTE recurrence, in cancer-associated thrombosis and in those with COVID-19. Therefore, given the known biological function of CRP in lifying inflammation and tissue damage, this raises the prospect that CRP may play a role in promoting VTE formation in the context of concurrent inflammation. However, further investigation is required to unravel whether CRP plays a direct role in the pathogenesis of VTE, the utility of which will be in developing novel prophylactic or therapeutic strategies to target thromboinflammation.
Publisher: Public Library of Science (PLoS)
Date: 27-03-2017
Publisher: American Chemical Society (ACS)
Date: 14-04-2014
DOI: 10.1021/ML500077J
Publisher: Springer Science and Business Media LLC
Date: 09-05-2004
DOI: 10.1038/NSB839
Publisher: American Society for Microbiology
Date: 30-04-2019
Abstract: The cholesterol-dependent cytolysins (CDCs) are the archetype for the superfamily of oligomeric pore-forming proteins that includes the membrane attack complex erforin (MACPF) family of immune defense proteins and the stonefish venom toxins (SNTX). The CDC/MACPF/SNTX family exhibits a common protein fold, which forms a membrane-spanning β-barrel pore. We show that changing the relative stability of an extensive intramolecular interface within this fold, which is necessarily disrupted to form the large β-barrel pore, dramatically alters the kinetic and temperature-dependent properties of CDC pore formation. These studies show that the CDCs and other members of the CDC/MACPF/SNTX superfamily have the capacity to significantly alter their pore-forming properties to function under widely different environmental conditions encountered by these species.
Publisher: Elsevier BV
Date: 09-2016
Publisher: Elsevier BV
Date: 07-1994
Publisher: Cold Spring Harbor Laboratory
Date: 19-10-2021
DOI: 10.1101/2021.10.19.464937
Abstract: The cholesterol-dependent cytolysin perfringolysin O (PFO) is secreted by Clostridium perfringens as a bacterial virulence factor able to form giant ring-shaped pores that perforate and ultimately lyse mammalian cell membranes. To resolve the kinetics of all steps in the assembly pathway, we have used single-molecule fluorescence imaging to follow the dynamics of PFO on dye-loaded liposomes that lead to opening of a pore and release of the encapsulated dye. Formation of a long-lived membrane-bound PFO dimer nucleates the growth of an irreversible oligomer. The growing oligomer can insert into the membrane and open a pore at stoichiometries ranging from tetramers to full rings (∼35-mers), whereby the rate of insertion increases linearly with the number of subunits. Oligomers that insert before the ring is complete continue to grow by monomer addition post insertion. Overall, our observations suggest that PFO membrane insertion is kinetically controlled.
Publisher: American Society of Hematology
Date: 09-08-2018
DOI: 10.1182/BLOODADVANCES.2018018820
Abstract: Novel Tfrc-mutant mouse identified in ENU mutagenesis screen with stable receptor expression. Flow imaging cytometry demonstrates microcytosis in mutants derives from dysfunctional receptor-mediated endocytosis of Tf-TfR complex.
Publisher: American Society for Microbiology
Date: 27-08-2019
Abstract: A unique feature of the CDC/MACPF/SNTX (cholesterol-dependent cytolysin/membrane attack complex perforin/stonefish toxin) superfamily of pore-forming toxins is that the β-strands that comprise the β-barrel pore are derived from a pair of α-helical bundles. These studies reveal the molecular basis by which the formation of intermolecular interactions within the prepore complex drive the disruption of intramolecular interactions within each monomer of the prepore to trigger the α-helical–to–β-strand transition and formation of the β-barrel pore.
Publisher: Elsevier BV
Date: 05-2019
DOI: 10.1016/J.HUMIMM.2018.11.002
Abstract: The oxytocinase subfamily of M1 aminopeptidases plays an important role in processing and trimming of peptides for presentation on major histocompatibility (MHC) Class I molecules. Several large-scale genomic studies have identified association of members of this family of enzymes, most notably ERAP1 and ERAP2, with immune-mediated diseases including ankylosing spondylitis, psoriasis and birdshot chorioretinopathy. Much is now known about the genetics of these enzymes and how genetic variants alter their function, but how these variants contribute to disease remains largely unresolved. Here we discuss what is known about their structure and function and highlight some of the knowledge gaps that affect development of drugs targeting these enzymes.
Publisher: Springer Science and Business Media LLC
Date: 21-11-2008
Publisher: Springer Science and Business Media LLC
Date: 05-08-2019
DOI: 10.1038/S41564-019-0524-4
Abstract: The activity of the proteasome 20S catalytic core is regulated by protein complexes that bind to one or both ends. The PA28 regulator stimulates 20S proteasome peptidase activity in vitro, but its role in vivo remains unclear. Here, we show that genetic deletion of the PA28 regulator from Plasmodium falciparum (Pf) renders malaria parasites more sensitive to the antimalarial drug dihydroartemisinin, indicating that PA28 may play a role in protection against proteotoxic stress. The crystal structure of PfPA28 reveals a bell-shaped molecule with an inner pore that has a strong segregation of charges. Small-angle X-ray scattering shows that disordered loops, which are not resolved in the crystal structure, extend from the PfPA28 heptamer and surround the pore. Using single particle cryo-electron microscopy, we solved the structure of Pf20S in complex with one and two regulatory PfPA28 caps at resolutions of 3.9 and 3.8 Å, respectively. PfPA28 binds Pf20S asymmetrically, strongly engaging subunits on only one side of the core. PfPA28 undergoes rigid body motions relative to Pf20S. Molecular dynamics simulations support conformational flexibility and a leaky interface. We propose lateral transfer of short peptides through the dynamic interface as a mechanism facilitating the release of proteasome degradation products.
Publisher: Oxford University Press (OUP)
Date: 11-2007
DOI: 10.1534/GENETICS.107.075838
Abstract: As classical phase II detoxification enzymes, glutathione S-transferases (GSTs) have been implicated in insecticide resistance and may have evolved in response to toxins in the niche-defining feeding substrates of Drosophila species. We have annotated the GST genes of the 12 Drosophila species with recently sequenced genomes and analyzed their molecular evolution. Gene copy number variation is attributable mainly to unequal crossing-over events in the large δ and ε clusters. Within these gene clusters there are also GST genes with slowly erging orthologs. This implies that they have their own unique functions or have spatial/temporal expression patterns that impose significant selective constraints. Searches for positively selected sites within the GSTs identified G171K in GSTD1, a protein that has previously been shown to be capable of metabolizing the insecticide DDT. We find that the same radical substitution (G171K) in the substrate-binding domain has occurred at least three times in the Drosophila radiation. Homology-modeling places site 171 distant from the active site but adjacent to an alternative DDT-binding site. We propose that the parallel evolution observed at this site is an adaptive response to an environmental toxin and that sequencing of historical alleles suggests that this toxin was not a synthetic insecticide.
Publisher: Elsevier BV
Date: 06-2010
DOI: 10.1016/J.JMB.2010.04.020
Abstract: GSTD1 is one of several insect glutathione S-transferases capable of metabolizing the insecticide DDT. Here we use crystallography and NMR to elucidate the binding of DDT and glutathione to GSTD1. The crystal structure of Drosophila melanogaster GSTD1 has been determined to 1.1 A resolution, which reveals that the enzyme adopts the canonical GST fold but with a partially occluded active site caused by the packing of a C-terminal helix against one wall of the binding site for substrates. This helix would need to unwind or be displaced to enable catalysis. When the C-terminal helix is removed from the model of the crystal structure, DDT can be computationally docked into the active site in an orientation favoring catalysis. Two-dimensional (1)H,(15)N heteronuclear single-quantum coherence NMR experiments of GSTD1 indicate that conformational changes occur upon glutathione and DDT binding and the residues that broaden upon DDT binding support the predicted binding site. We also show that the ancestral GSTD1 is likely to have possessed DDT dehydrochlorinase activity because both GSTD1 from D. melanogaster and its sibling species, Drosophila simulans, have this activity.
Publisher: Elsevier BV
Date: 30-05-2008
Publisher: Wiley
Date: 02-2010
DOI: 10.1002/ANA.21849
Publisher: American Chemical Society (ACS)
Date: 09-03-2011
DOI: 10.1021/BI101893W
Abstract: The development of inhibitors of insulin-regulated aminopeptidase (IRAP), a membrane-bound zinc metallopeptidase, is a promising approach for the discovery of drugs for the treatment of memory loss such as that associated with Alzheimer's disease. There is, however, no consensus in the literature about the mechanism by which inhibition occurs. Sequence alignments, secondary structure predictions, and homology models based on the structures of recently determined related metallopeptidases suggest that the extracellular region consists of four domains. Partial proteolysis and mass spectrometry reported here confirm some of the domain boundaries. We have produced purified recombinant fragments of human IRAP on the basis of these data and examined their kinetic and biochemical properties. Full-length extracellular constructs assemble as dimers with different nonoverlapping fragments dimerizing as well, suggesting an extended dimer interface. Only recombinant fragments containing domains 1 and 2 possess aminopeptidase activity and bind the radiolabeled hexapeptide inhibitor, angiotensin IV (Ang IV). However, fragments lacking domains 3 and 4 possess reduced activity, although they still bind a range of inhibitors with the same affinity as longer fragments. In the presence of Ang IV, IRAP is resistant to proteolysis, suggesting significant conformational changes occur upon binding of the inhibitor. We show that IRAP has a second Zn(2+) binding site, not associated with the catalytic region, which is lost upon binding Ang IV. Modulation of activity caused by domains 3 and 4 is consistent with a conformational change regulating access to the active site of IRAP.
Publisher: Wiley
Date: 04-05-2009
Abstract: Double trouble: A hybrid organic-inorganic (organometallic) inhibitor was designed to target glutathione transferases. The metal center is used to direct protein binding, while the organic moiety acts as the active-site inhibitor (see picture). The mechanism of inhibition was studied using a range of biophysical and biochemical methods.
Publisher: American Chemical Society (ACS)
Date: 17-06-2019
DOI: 10.1021/ACS.CHEMREV.9B00090
Abstract: The cholesterol-dependent cytolysins (CDCs) are a family of bacterial protein toxins specifically targeting eukaryotic cells through the absolute requirement for high concentrations of cholesterol in the target cells' lipid membrane. The soluble monomeric protein secreted by the bacteria oligomerizes on the surface of the target cell, and the complex formed then undergoes a concerted structural transition that results in the creation of a multimeric protein pore. Recognition of the cholesterol-rich membrane by CDCs is a surprisingly subtle process that takes place at the interface between the membrane and surrounding aqueous environment. The structure and composition of the lipid membrane modulates the efficiency with which the protein can identify cholesterol and alters the concentration of sterol required for membrane binding. Some of the details of the interplay between protein and membrane remain to be resolved, and in this review we present a current perspective on CDC pore formation, with particular focus on the role of the lipid bilayer and cholesterol accessibility.
Publisher: Elsevier BV
Date: 08-2016
DOI: 10.1016/J.JACI.2016.03.025
Abstract: The selection of pharmacotherapy for patients with allergic rhinitis (AR) depends on several factors, including age, prominent symptoms, symptom severity, control of AR, patient preferences, and cost. Allergen exposure and the resulting symptoms vary, and treatment adjustment is required. Clinical decision support systems (CDSSs) might be beneficial for the assessment of disease control. CDSSs should be based on the best evidence and algorithms to aid patients and health care professionals to jointly determine treatment and its step-up or step-down strategy depending on AR control. Contre les MAladies Chroniques pour un VIeillissement Actif en Languedoc-Roussillon (MACVIA-LR [fighting chronic diseases for active and healthy ageing]), one of the reference sites of the European Innovation Partnership on Active and Healthy Ageing, has initiated an allergy sentinel network (the MACVIA-ARIA Sentinel Network). A CDSS is currently being developed to optimize AR control. An algorithm developed by consensus is presented in this article. This algorithm should be confirmed by appropriate trials.
Publisher: Springer Science and Business Media LLC
Date: 28-03-2006
Publisher: Elsevier BV
Date: 02-2015
DOI: 10.1016/J.BMCL.2014.11.024
Abstract: Respiratory syncytial virus (RSV) is a major cause of respiratory tract infections in infants, young children and adults. 1,2,3,9b-Tetrahydro-5H-imidazo[2,1-a]isoindol-5-ones with general structure 1 were previously identified as promising inhibitors of RSV targeting the fusion glycoprotein. In particular, the introduction of a nitrogen at the 8-position of the tricyclic core yielded lead compounds 2 and 3. Extensive exploration of the R(2) group established that certain heterocyclic amides conferred potent RSV A&B activity and a good balance of physicochemical and pharmacokinetic properties. The antiviral activity was found to reside in a single enantiomer and compound 33a, (9bS)-9b-(4-chlorophenyl)-1-(pyridin-3-ylcarbonyl)-1,2,3,9b-tetrahydro-5H-imidazo[1',2':1,2]pyrrolo[3,4-c]pyridin-5-one (known as BTA9881), was identified as a candidate for preclinical development.
Publisher: Elsevier BV
Date: 08-2007
Publisher: International Union of Crystallography (IUCr)
Date: 26-11-2015
DOI: 10.1107/S1399004715018519
Abstract: Pseudomonas aeruginosa is an opportunistic human pathogen for which new antimicrobial drug options are urgently sought. P. aeruginosa disulfide-bond protein A1 (PaDsbA1) plays a pivotal role in catalyzing the oxidative folding of multiple virulence proteins and as such holds great promise as a drug target. As part of a fragment-based lead discovery approach to PaDsbA1 inhibitor development, the identification of a crystal form of PaDsbA1 that was more suitable for fragment-soaking experiments was sought. A previously identified crystallization condition for this protein was unsuitable, as in this crystal form of PaDsbA1 the active-site surface loops are engaged in the crystal packing, occluding access to the target site. A single residue involved in crystal-packing interactions was substituted with an amino acid commonly found at this position in closely related enzymes, and this variant was successfully used to generate a new crystal form of PaDsbA1 in which the active-site surface is more accessible for soaking experiments. The PaDsbA1 variant displays identical redox character and in vitro activity to wild-type PaDsbA1 and is structurally highly similar. Two crystal structures of the PaDsbA1 variant were determined in complex with small molecules bound to the protein active site. These small molecules (MES, glycerol and ethylene glycol) were derived from the crystallization or cryoprotectant solutions and provide a proof of principle that the reported crystal form will be amenable to co-crystallization and soaking with small molecules designed to target the protein active-site surface.
Publisher: eLife Sciences Publications, Ltd
Date: 24-08-2022
DOI: 10.7554/ELIFE.74901
Abstract: The cholesterol-dependent cytolysin perfringolysin O (PFO) is secreted by Clostridium perfringens as a bacterial virulence factor able to form giant ring-shaped pores that perforate and ultimately lyse mammalian cell membranes. To resolve the kinetics of all steps in the assembly pathway, we have used single-molecule fluorescence imaging to follow the dynamics of PFO on dye-loaded liposomes that lead to opening of a pore and release of the encapsulated dye. Formation of a long-lived membrane-bound PFO dimer nucleates the growth of an irreversible oligomer. The growing oligomer can insert into the membrane and open a pore at stoichiometries ranging from tetramers to full rings (~35 mers), whereby the rate of insertion increases linearly with the number of subunits. Oligomers that insert before the ring is complete continue to grow by monomer addition post insertion. Overall, our observations suggest that PFO membrane insertion is kinetically controlled.
Publisher: Bentham Science Publishers Ltd.
Date: 11-2007
DOI: 10.2174/092986707782360097
Abstract: A successful unified pharmacophore/receptor model which has guided the synthesis of subtype selective compounds is reviewed in light of recent developments both in ligand synthesis and structural studies of the binding site itself. The evaluation of experimental data in combination with a comparative model of the alpha1beta2gamma2 GABA(A) receptor leads to an orientation of the pharmacophore model within the Bz BS. Results not only are important for the rational design of selective ligands, but also for the identification and evaluation of possible roles which specific residues may have within the benzodiazepine binding pocket.
Publisher: Wiley
Date: 12-2008
Publisher: Wiley
Date: 2004
DOI: 10.1110/PS.03340004
Abstract: AMP-activated protein kinase (AMPK) is a alphabetagamma heterotrimer that is activated in response to both hormones and intracellular metabolic stress signals. AMPK is regulated by phosphorylation on the alpha subunit and by AMP allosteric control previously thought to be mediated by both alpha and gamma subunits. Here we present evidence that adjacent gamma subunit pairs of CBS repeat sequences (after Cystathionine Beta Synthase) form an AMP binding site related to, but distinct from the classical AMP binding site in phosphorylase, that can also bind ATP. The AMP binding site of the gamma(1) CBS1/CBS2 pair, modeled on the structures of the CBS sequences present in the inosine monophosphate dehydrogenase crystal structure, contains three arginine residues 70, 152, and 171 and His151. The yeast gamma homolog, snf4 contains a His151Gly substitution, and when this is introduced into gamma(1), AMP allosteric control is substantially lost and explains why the yeast snf1p/snf4p complex is insensitive to AMP. Arg70 in gamma(1) corresponds to the site of mutation in human gamma(2) and pig gamma(3) genes previously identified to cause an unusual cardiac phenotype and glycogen storage disease, respectively. Mutation of any of AMP binding site Arg residues to Gln substantially abolishes AMP allosteric control in expressed AMPK holoenzyme. The Arg/Gln mutations also suppress the previously described inhibitory properties of ATP and render the enzyme constitutively active. We propose that ATP acts as an intrasteric inhibitor by bridging the alpha and gamma subunits and that AMP functions to derepress AMPK activity.
Publisher: Elsevier BV
Date: 04-2007
Publisher: Cold Spring Harbor Laboratory
Date: 11-2020
DOI: 10.1101/2020.11.01.364042
Abstract: The ubiquitous CLC protein superfamily consists of channels, that permit passive diffusion of Cl ions across biological membranes, and pumps, that can actively transport Cl ions against their electrochemical gradient yet, puzzlingly, both types share a strongly conserved Cl ion transport pathway comprised of three consecutive binding sites. This raises the question how does the same pathway support passive diffusion in CLC channels and active transport in CLC pumps? Based on high-resolution structural data current theories suggest that subtle structural differences in the conserved pathway allow CLC channels to ‘leak’ Cl ions. A recent cryo-electron microscopy structure of the human ClC-1 channel does not show occupancy of the central Cl ion binding site but reveals a wide intracellular vestibule that bifurcates from the conserved pathway in this region. Here we show that replacing residues that line the ClC-1 intracellular vestibule with the corresponding residues of CLC pumps resulted in interactions between permeating anions at neighbouring binding sites and altered anion selectivity. Removing the side chain of a strictly conserved tyrosine residue, that coordinates Cl ion at the central binding site of CLC pumps, removed multi-ion behaviour in ClC-1 mutants. In contrast, removing the side chain of a highly conserved glutamate residue that transiently occupies Cl ion binding sites, as part of the transport mechanism of CLC pumps and the mechanism that opens and closes CLC channels, only partially removed multi-ion behaviour in ClC-1 mutants. Our findings show that structural differences between CLC channels and pumps, outside of the conserved Cl ion transport pathway, fundamentally affect anion permeation in ClC-1 channels. Some CLC proteins are passive Cl - channels while others are active Cl - pumps but, paradoxically, both share a conserved, canonical, Cl - permeation pathway. Here Bennetts, Morton and Parker show that ‘pump-like’ mutations in a poorly conserved region, located remotely from the canonical pathway, affect anion permeation in human ClC-1 channels.
Publisher: Canadian Science Publishing
Date: 04-2008
DOI: 10.1139/O08-037
Abstract: Inhibition of insulin-regulated aminopeptidase (IRAP) has been demonstrated to facilitate memory in rodents, making IRAP a potential target for the development of cognitive enhancing therapies. In this study, we generated a 3-D model of the catalytic domain of IRAP based on the crystal structure of leukotriene A4 hydrolase (LTA4H). This model identified two key residues at the ‘entrance’ of the catalytic cleft of IRAP, Ala427 and Leu483, which present a more open arrangement of the S1 subsite compared with LTA4H. These residues may define the size and 3-D structure of the catalytic pocket, thereby conferring substrate and inhibitor specificity. Alteration of the S1 subsite by the mutation A427Y in IRAP markedly increased the rate of substrate cleavage V of the enzyme for a synthetic substrate, although a corresponding increase in the rate of cleavage of peptide substrates Leu-enkephalin and vasopressin was was not apparent. In contrast, [L483F]IRAP demonstrated a 30-fold decrease in activity due to changes in both substrate affinity and rate of substrate cleavage. [L483F]IRAP, although capable of efficiently cleaving the N-terminal cysteine from vasopressin, was unable to cleave the tyrosine residue from either Leu-enkephalin or Cyt6-desCys 1 -vasopressin (2–9), both substrates of IRAP. An 11-fold reduction in the affinity of the peptide inhibitor norleucine 1 -angiotensin IV was observed, whereas the affinity of angiotensin IV remained unaltered. In additionm we predict that the peptide inhibitors bind to the catalytic site, with the NH 2 -terminal P1 residue occupying the catalytic cleft (S1 subsite) in a manner similar to that proposed for peptide substrates.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 03-06-2022
Abstract: Aminoacyl transfer RNA (tRNA) synthetases (aaRSs) are attractive drug targets, and we present class I and II aaRSs as previously unrecognized targets for adenosine 5′-monophosphate–mimicking nucleoside sulfamates. The target enzyme catalyzes the formation of an inhibitory amino acid–sulfamate conjugate through a reaction-hijacking mechanism. We identified adenosine 5′-sulfamate as a broad-specificity compound that hijacks a range of aaRSs and ML901 as a specific reagent a specific reagent that hijacks a single aaRS in the malaria parasite Plasmodium falciparum , namely tyrosine RS ( Pf YRS). ML901 exerts whole-life-cycle–killing activity with low nanomolar potency and single-dose efficacy in a mouse model of malaria. X-ray crystallographic studies of plasmodium and human YRSs reveal differential flexibility of a loop over the catalytic site that underpins differential susceptibility to reaction hijacking by ML901.
Location: Australia
Start Date: 01-2020
End Date: 2023
Amount: $560,000.00
Funder: Australian Research Council
View Funded ActivityStart Date: 07-2003
End Date: 12-2008
Amount: $225,000.00
Funder: Australian Research Council
View Funded ActivityStart Date: 03-2010
End Date: 12-2012
Amount: $600,000.00
Funder: Australian Research Council
View Funded Activity