ORCID Profile
0000-0002-3518-9785
Current Organisations
National Institute of Immunology
,
Monash University
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Enzymes | Characterisation of Biological Macromolecules | Biochemistry and Cell Biology | Structural Biology (incl. Macromolecular Modelling) |
Expanding Knowledge in the Biological Sciences | Infectious Diseases
Publisher: Worldwide Protein Data Bank
Date: 02-12-2008
DOI: 10.2210/PDB3BI8/PDB
Publisher: Elsevier BV
Date: 09-2012
DOI: 10.1016/J.JMB.2012.06.006
Abstract: The malarial aminopeptidases have emerged as promising new drug targets for the development of novel antimalarial drugs. The M18AAP of Plasmodium falciparum malaria is a metallo-aminopeptidase that we show demonstrates a highly restricted specificity for peptides with an N-terminal Glu or Asp residue. Thus, the enzyme may function alongside other aminopeptidases in effecting the complete degradation or turnover of proteins, such as host hemoglobin, which provides a free amino acid pool for the growing parasite. Inhibition of PfM18AAP's function using antisense RNA is detrimental to the intra-erythrocytic malaria parasite and, hence, it has been proposed as a potential novel drug target. We report the X-ray crystal structure of the PfM18AAP aminopeptidase and reveal its complex dodecameric assembly arranged via dimer and trimer units that interact to form a large tetrahedron shape that completely encloses the 12 active sites within a central cavity. The four entry points to the catalytic lumen are each guarded by 12 large flexible loops that could control substrate entry into the catalytic sites. PfM18AAP thus resembles a proteasomal-like machine with multiple active sites able to degrade peptide substrates that enter the central lumen. The Plasmodium enzyme shows significant structural differences around the active site when compared to recently determined structures of its mammalian and human homologs, which provides a platform from which a rational approach to inhibitor design of new malaria-specific drugs can begin.
Publisher: International Union of Crystallography (IUCr)
Date: 14-02-2009
Publisher: Worldwide Protein Data Bank
Date: 04-12-2013
DOI: 10.2210/PDB4I7V/PDB
Publisher: Worldwide Protein Data Bank
Date: 04-12-2013
DOI: 10.2210/PDB4I7U/PDB
Publisher: Springer Science and Business Media LLC
Date: 15-06-2015
DOI: 10.1038/NSMB.3048
Publisher: Public Library of Science (PLoS)
Date: 05-07-2012
Publisher: Worldwide Protein Data Bank
Date: 17-06-2015
DOI: 10.2210/PDB4WZ9/PDB
Publisher: MDPI AG
Date: 23-08-2021
DOI: 10.3390/IJMS22169094
Abstract: The cytoplasmic retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) initiate interferon (IFN) production and antiviral gene expression in response to RNA virus infection. Consequently, RLR signalling is tightly regulated by both host and viral factors. Tripartite motif protein 25 (TRIM25) is an E3 ligase that ubiquitinates multiple substrates within the RLR signalling cascade, playing both ubiquitination-dependent and -independent roles in RIG-I-mediated IFN induction. However, additional regulatory roles are emerging. Here, we show a novel interaction between TRIM25 and another protein in the RLR pathway that is essential for type I IFN induction, DEAD-box helicase 3X (DDX3X). In vitro assays and knockdown studies reveal that TRIM25 ubiquitinates DDX3X at lysine 55 (K55) and that TRIM25 and DDX3X cooperatively enhance IFNB1 induction following RIG-I activation, but the latter is independent of TRIM25’s catalytic activity. Furthermore, we found that the influenza A virus non-structural protein 1 (NS1) disrupts the TRIM25:DDX3X interaction, abrogating both TRIM25-mediated ubiquitination of DDX3X and cooperative activation of the IFNB1 promoter. Thus, our results reveal a new interplay between two RLR-host proteins that cooperatively enhance IFN-β production. We also uncover a new and further mechanism by which influenza A virus NS1 suppresses host antiviral defence.
Publisher: Worldwide Protein Data Bank
Date: 04-12-2013
DOI: 10.2210/PDB4I7W/PDB
Publisher: MDPI AG
Date: 24-03-2019
DOI: 10.3390/CELLS8030281
Abstract: Dengue virus (DENV) threatens almost 70% of the world’s population, with no effective vaccine or therapeutic currently available. A key contributor to infection is nuclear localisation in the infected cell of DENV nonstructural protein 5 (NS5) through the action of the host importin (IMP) α/β1 proteins. Here, we used a range of microscopic, virological and biochemical/biophysical approaches to show for the first time that the small molecule GW5074 has anti-DENV action through its novel ability to inhibit NS5–IMPα/β1 interaction in vitro as well as NS5 nuclear localisation in infected cells. Strikingly, GW5074 not only inhibits IMPα binding to IMPβ1, but can dissociate preformed IMPα/β1 heterodimer, through targeting the IMPα armadillo (ARM) repeat domain to impact IMPα thermal stability and α-helicity, as shown using analytical ultracentrifugation, thermostability analysis and circular dichroism measurements. Importantly, GW5074 has strong antiviral activity at low µM concentrations against not only DENV-2, but also zika virus and West Nile virus. This work highlights DENV NS5 nuclear targeting as a viable target for anti-flaviviral therapeutics.
Publisher: Elsevier BV
Date: 02-2010
Publisher: Elsevier BV
Date: 09-2020
Publisher: International Union of Crystallography (IUCr)
Date: 27-02-2013
Publisher: Worldwide Protein Data Bank
Date: 25-07-2012
DOI: 10.2210/PDB3TUU/PDB
Publisher: Elsevier BV
Date: 10-2008
Publisher: Wiley
Date: 11-06-2014
DOI: 10.1002/PROT.24539
Abstract: Agrobacterium tumefaciens is a Gram-negative soil-borne bacterium that causes Crown Gall disease in many economically important crops. The absence of a suitable chemical treatment means there is a need to discover new anti-Crown Gall agents and also characterize bona fide drug targets. One such target is dihydrodipicolinate synthase (DHDPS), a homo-tetrameric enzyme that catalyzes the committed step in the metabolic pathway yielding meso-diaminopimelate and lysine. Interestingly, there are 10 putative DHDPS genes annotated in the A. tumefaciens genome, including three whose structures have recently been determined (PDB IDs: 3B4U, 2HMC, and 2R8W). However, we show using quantitative enzyme kinetic assays that nine of the 10 dapA gene products, including 3B4U, 2HMC, and 2R8W, lack DHDPS function in vitro. A sequence alignment showed that the product of the dapA7 gene contains all of the conserved residues known to be important for DHDPS catalysis and allostery. This gene was cloned and the recombinant product expressed and purified. Our studies show that the purified enzyme (i) possesses DHDPS enzyme activity, (ii) is allosterically inhibited by lysine, and (iii) adopts the canonical homo-tetrameric structure in both solution and the crystal state. This study describes for the first time the structure, function and allostery of the bona fide DHDPS from A. tumefaciens, which offers insight into the rational design of pesticide agents for combating Crown Gall disease.
Publisher: Worldwide Protein Data Bank
Date: 30-06-2010
DOI: 10.2210/PDB3A5F/PDB
Publisher: International Union of Crystallography (IUCr)
Date: 29-02-2008
Publisher: International Union of Crystallography (IUCr)
Date: 30-08-2012
Publisher: MDPI AG
Date: 30-09-2019
DOI: 10.3390/CELLS8101181
Abstract: DEAD-box helicase 3, X-linked (DDX3X) regulates the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR)-mediated antiviral response, but can also be a host factor contributing to the replication of viruses of significance to human health, such as human immunodeficiency virus type 1 (HIV-1). These roles are mediated in part through its ability to actively shuttle between the nucleus and the cytoplasm to modulate gene expression, although the trafficking mechanisms, and impact thereof on immune signaling and viral infection, are incompletely defined. We confirm that DDX3X nuclear export is mediated by the nuclear transporter exportin-1/CRM1, dependent on an N-terminal, leucine-rich nuclear export signal (NES) and the monomeric guanine nucleotide binding protein Ran in activated GTP-bound form. Transcriptome profiling and ELISA show that exportin-1-dependent export of DDX3X to the cytoplasm strongly impacts IFN-β production and the upregulation of immune genes in response to infection. That this is key to DDX3X’s antiviral role was indicated by enhanced infection by human parainfluenza virus-3 (hPIV-3)/elevated virus production when the DDX3X NES was inactivated. Our results highlight a link between nucleocytoplasmic distribution of DDX3X and its role in antiviral immunity, with strong relevance to hPIV-3, as well as other viruses such as HIV-1.
Publisher: Elsevier BV
Date: 2018
DOI: 10.1016/J.IJBIOMAC.2017.08.071
Abstract: Molecular self-assembly of biodegradable hiphilic polymers allows rational design of biocompatible nanomaterials for drug delivery. Use of substituted polysaccharides for such applications offers the ease of design and synthesis, and provides higher biofunctionality and biocompatibility to nanomaterials. The present work focuses on the synthesis, characterization and potential biomedical applications of self-assembled polysaccharide-based materials. We demonstrated that the synthesized hiphilic inulin self-assembled in aqueous medium into nanostructures with average size in the range of 146-486nm and encapsulated hydrophobic therapeutic molecule, ornidazole. Hydrophophic dehydropeptide was conjugated with inulin via a biocompatible ester linkage. Dehydrophenylalanine, an unusual amino acid, was incorporated in the peptide to make it stable at a broader range of pH as well as against proteases. The resulting core-shell type of nanostructures could encapsulate ornidazole in the hydrophobic core and released it in a controlled fashion. By taking the advantage of inulin, which gets degraded in the colon by colonic bacteria, the effect of enzyme, inulinase, present in the microflora of the large intestine, on inulin-peptide degradation followed by drug release has been studied. Altogether, small peptide conjugated to inulin offers novel scaffold for the future design of nanostructures with potential applications in the field of targeted drug delivery.
Publisher: Worldwide Protein Data Bank
Date: 22-09-2009
DOI: 10.2210/PDB3IRD/PDB
Publisher: Worldwide Protein Data Bank
Date: 04-09-2013
DOI: 10.2210/PDB4HNN/PDB
Publisher: MDPI AG
Date: 09-01-2020
DOI: 10.3390/CELLS9010170
Abstract: Viral disease is one of the greatest burdens for human health worldwide, with an urgent need for efficacious antiviral strategies. While antiviral drugs are available, in many cases, they are prone to the development of drug resistance. A way to overcome drug resistance associated with common antiviral therapies is to develop antivirals targeting host cellular co-factors critical to viral replication, such as DEAD-box helicase 3 X-linked (DDX3X), which plays key roles in RNA metabolism and the antiviral response. Here, we use biochemical/biophysical approaches and infectious assays to show for the first time that the small molecule RK-33 has broad-spectrum antiviral action by inhibiting the enzymatic activities of DDX3X. Importantly, we show that RK-33 is efficacious at low micromolar concentrations in limiting infection by human parainfluenza virus type 3 (hPIV-3), respiratory syncytial virus (RSV), dengue virus (DENV), Zika virus (ZIKV) or West Nile virus (WNV)—for all of which, no Food and Drug Administration (FDA)-approved therapeutic is widely available. These findings establish for the first time that RK-33 is a broad-spectrum antiviral agent that blocks DDX3X’s catalytic activities in vitro and limits viral replication in cells.
Publisher: International Union of Crystallography (IUCr)
Date: 24-03-2016
DOI: 10.1107/S205979831600142X
Abstract: Pyruvate kinase is a key regulatory enzyme involved in the glycolytic pathway. The crystal structure of Escherichia coli type I pyruvate kinase was first solved in 1995 at 2.5 Å resolution. However, the space group was ambiguous, being either primitive orthorhombic ( P 2 1 2 1 2 1 ) or C -centred orthorhombic ( C 222 1 ). Here, the structure determination and refinement of E. coli type I pyruvate kinase to 2.28 Å resolution are presented. Using the same crystallization conditions as reported previously, the enzyme was found to crystallize in space group P 2 1 . Determination of the space group was complicated owing to anisotropic data, pseudo-translational noncrystallographic symmetry and the pseudo-merohedrally twinned nature of the crystal, which was found to have very close to 50% twinning, leading to apparent orthorhombic symmetry and absences that were not inconsistent with P 2 1 2 1 2 1 . The unit cell contained two tetramers in the asymmetric unit (3720 residues) and, when compared with the orthorhombic structure, virtually all of the residues could be easily modelled into the density. Averaging of reflections into the lower symmetry space group with twinning provided tidier electron density that allowed ∼30 missing residues of the lid domain to be modelled for the first time. Moreover, residues in a flexible loop could be modelled and sulfate molecules are found in the allosteric binding domain, identifying the pocket that binds the allosteric activator fructose 1,6-bisphosphate in this isozyme for the first time. Lastly, we note the pedagogical benefits of difficult structures to emerging crystallographers.
Publisher: MDPI AG
Date: 10-12-2020
DOI: 10.3390/CELLS9122654
Abstract: The transport of host proteins into and out of the nucleus is key to host function. However, nuclear transport is restricted by nuclear pores that perforate the nuclear envelope. Protein intrinsic disorder is an inherent feature of this selective transport barrier and is also a feature of the nuclear transport receptors that facilitate the active nuclear transport of cargo, and the nuclear transport signals on the cargo itself. Furthermore, intrinsic disorder is an inherent feature of viral proteins and viral strategies to disrupt host nucleocytoplasmic transport to benefit their replication. In this review, we highlight the role that intrinsic disorder plays in the nuclear transport of host and viral proteins. We also describe viral subversion mechanisms of the host nuclear transport machinery in which intrinsic disorder is a feature. Finally, we discuss nuclear import and export as therapeutic targets for viral infectious disease.
Publisher: Springer Science and Business Media LLC
Date: 10-01-2018
DOI: 10.1038/S41598-017-18742-8
Abstract: Hendra virus (HeV) is a paramyxovirus that causes lethal disease in humans, for which no vaccine or antiviral agent is available. HeV V protein is central to pathogenesis through its ability to interact with cytoplasmic host proteins, playing key antiviral roles. Here we use immunoprecipitation, siRNA knockdown and confocal laser scanning microscopy to show that HeV V shuttles to and from the nucleus through specific host nuclear transporters. Spectroscopic and small angle X-ray scattering studies reveal HeV V undergoes a disorder-to-order transition upon binding to either importin α/β1 or exportin-1/Ran-GTP, dependent on the V N-terminus. Importantly, we show that specific inhibitors of nuclear transport prevent interaction with host transporters, and reduce HeV infection. These findings emphasize the critical role of host-virus interactions in HeV infection, and potential use of compounds targeting nuclear transport, such as the FDA-approved agent ivermectin, as anti-HeV agents.
Publisher: Public Library of Science (PLoS)
Date: 25-06-2012
Publisher: Elsevier BV
Date: 07-2018
DOI: 10.1016/J.STR.2018.04.014
Abstract: Protein dynamics manifested through structural flexibility play a central role in the function of biological molecules. Here we explore the substrate-mediated change in protein flexibility of an antibiotic target enzyme, Clostridium botulinum dihydrodipicolinate synthase. We demonstrate that the substrate, pyruvate, stabilizes the more active dimer-of-dimers or tetrameric form. Surprisingly, there is little difference between the crystal structures of apo and substrate-bound enzyme, suggesting protein dynamics may be important. Neutron and small-angle X-ray scattering experiments were used to probe substrate-induced dynamics on the sub-second timescale, but no significant changes were observed. We therefore developed a simple technique, coined protein dynamics-mass spectrometry (ProD-MS), which enables measurement of time-dependent alkylation of cysteine residues. ProD-MS together with X-ray crystallography and analytical ultracentrifugation analyses indicates that pyruvate locks the conformation of the dimer that promotes docking to the more active tetrameric form, offering insight into ligand-mediated stabilization of multimeric enzymes.
Publisher: Elsevier BV
Date: 2021
Publisher: Springer Science and Business Media LLC
Date: 26-01-2013
DOI: 10.1007/S11103-013-0014-7
Abstract: Lysine is one of the most limiting amino acids in plants and its biosynthesis is carefully regulated through inhibition of the first committed step in the pathway catalyzed by dihydrodipicolinate synthase (DHDPS). This is mediated via a feedback mechanism involving the binding of lysine to the allosteric cleft of DHDPS. However, the precise allosteric mechanism is yet to be defined. We present a thorough enzyme kinetic and thermodynamic analysis of lysine inhibition of DHDPS from the common grapevine, Vitis vinifera (Vv). Our studies demonstrate that lysine binding is both tight (relative to bacterial DHDPS orthologs) and cooperative. The crystal structure of the enzyme bound to lysine (2.4 Å) identifies the allosteric binding site and clearly shows a conformational change of several residues within the allosteric and active sites. Molecular dynamics simulations comparing the lysine-bound (PDB ID 4HNN) and lysine free (PDB ID 3TUU) structures show that Tyr132, a key catalytic site residue, undergoes significant rotational motion upon lysine binding. This suggests proton relay through the catalytic triad is attenuated in the presence of lysine. Our study reveals for the first time the structural mechanism for allosteric inhibition of DHDPS from the common grapevine.
Publisher: International Union of Crystallography (IUCr)
Date: 25-11-2011
DOI: 10.1107/S1744309111038395
Abstract: Dihydrodipicolinate synthase (DHDPS) catalyses the first committed step of the lysine-biosynthesis pathway in bacteria, plants and some fungi. This study describes the cloning, expression, purification and crystallization of DHDPS from the grapevine Vitis vinifera (Vv-DHDPS). Following in-drop cleavage of the hexahistidine tag, cocrystals of Vv-DHDPS with the substrate pyruvate were grown in 0.1 M Bis-Tris propane pH 8.2, 0.2 M sodium bromide, 20%( w / v ) PEG 3350. X-ray diffraction data in space group P 1 at a resolution of 2.2 Å are presented. Preliminary diffraction data analysis indicated the presence of eight molecules per asymmetric unit ( V M = 2.55 Å 3 Da −1 , 52% solvent content). The pending crystal structure of Vv-DHDPS will provide insight into the molecular evolution in quaternary structure of DHDPS enzymes.
Start Date: 2019
End Date: 09-2020
Amount: $416,092.00
Funder: Australian Research Council
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