6 results for Arcus, VL

  • The potential impact of structural genomics on tuberculosis drug discovery

    Arcus, VL; Lott, Jeremy; Johnston, JM; Baker, Edward (2006-01)

    Journal article
    The University of Auckland Library

    Mycobacterium tuberculosis, the causative agent of tuberculosis (TB) in humans, is a devastating infectious organism that kills approximately two million people annually. The current suite of antibiotics used to treat TB faces two main difficulties: (i) the emergence of multidrug-resistant (MDR) strains of M. tuberculosis, and (ii) the persistent state of the bacterium, which is less susceptible to antibiotics and causes very long antibiotic treatment regimes. The complete genome sequences of a laboratory strain (H37Rv) and a clinical strain (CDC1551) of M. tuberculosis and the concurrent identification of all the open reading frames that encode proteins within this organism, present structural biologists with a wide array of protein targets for structure determination. Comparative genomics of the species that make up the M. tuberculosis complex has also added an array of genomic information to our understanding of these organisms. In response to this, structural genomics consortia have been established for targeting proteins from M. tuberculosis. This review looks at the progress of these major initiatives and the potential impact of large scale structure determination efforts on the development of inhibitors to many proteins. Increasing sophistication in structure-based drug design approaches, in combination with increasing numbers of protein structures and inhibitors for TB proteins, will have a significant impact on the downstream development of TB antibiotics.

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  • The multi-functional histone-like Lsr2 protects mycobacteria against reactive oxygen intermediates

    Colangeli, R; Haq, A; Arcus, VL; Summers, Emma; McBride, Andrew; Mitra, Alok; Radjainia, Mazdak; Khjao, A; jacobs, WR; Salgame, P; Allande, D (2009)

    Journal article
    The University of Auckland Library

    Mycobacterium tuberculosis has evolved a number of strategies to survive within the hostile environment of host phagocytes. Reactive nitrogen and oxygen intermediates (RNI and ROI) are among the most effective antimycobacterial molecules generated by the host during infection. Lsr2 is a M. tuberculosis protein with histone-like features, including the ability to regulate a variety of transcriptional responses in mycobacteria. Here we demonstrate that Lsr2 protects mycobacteria against ROI in vitro and during macrophage infection. Furthermore, using macrophages derived from NOS−/− and Phox−/− mice, we demonstrate that Lsr2 is important in protecting against ROI but not RNI. The protection provided by Lsr2 protein is not the result of its ability to either bind iron or scavenge hydroxyl radicals. Instead, electron microscopy and DNA-binding studies suggest that Lsr2 shields DNA from reactive intermediates by binding bacterial DNA and physically protecting it. Thus, Lsr2 appears to be a unique protein with both histone-like properties and protective features that may be central to M. tuberculosis pathogenesis. In addition, evidence indicates that lsr2 is an essential gene in M. tuberculosis. Because of its essentiality, Lsr2 may represent an excellent candidate as a drug target.

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  • Expression and purification of an adenylation domain from a eukaryotic nonribosomal peptide synthetase: Using structural genomics tools for a challenging target

    Lee, TV; Lott, JS; Johnson, RD; Arcus, VL (2010)

    Journal article
    The University of Auckland Library

    Nonribosomal peptide synthetases (NRPSs) are large multimodular and multidomain enzymes that are involved in synthesising an array of molecules that are important in human and animal health. NRPSs are found in both bacteria and fungi but most of the research to date has focused on the bacterial enzymes. This is largely due to the technical challenges in producing active fungal NRPSs, which stem from their large size and multidomain nature. In order to target fungal NRPS domains for biochemical and structural characterisation, we tackled this challenge by using the cloning and expression tools of structural genomics to screen the many variables that can influence the expression and purification of proteins. Using these tools we have screened 32 constructs containing 16 different fungal NRPS domains or domain combinations for expression and solubility. Two of these yielded soluble protein with one, the third adenylation domain of the SidN NRPS (SidNA3) from the grass endophyte Neotyphodium lolii, being tractable for purification using Ni-affinity resin. The initial purified protein exhibited poor solution behaviour but optimisation of the expression construct and the buffer conditions used for purification, resulted in stable recombinant protein suitable for biochemical characterisation, crystallisation and structure determination.

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  • Structure of a Eukaryotic Nonribosomal Peptide Synthetase Adenylation Domain That Activates a Large Hydroxamate Amino Acid in Siderophore Biosynthesis

    Lee, Tet; Johnson, LJ; Johnson, RD; Koulman, A; Lane, GA; Lott, Jeremy; Arcus, VL (2010)

    Journal article
    The University of Auckland Library

    Non-ribosomal peptide synthetases (NRPSs) are large, multi-domain proteins that are involved in the biosynthesis of an array of secondary metabolites. We report the structure of the third adenylation domain from the siderophore-synthesizing NRPS, SidN, from the endophytic fungus, Neotyphodium lolii. This is the first structure of a eukaryotic NRPS domain and it reveals a large binding pocket required to accommodate the unusual amino acid substrate, N´-cis-anhydromevalonyl-N´-hydroxy-L-ornithine (cis-AMHO). The specific activation of cis-AMHO was confirmed biochemically and an AMHO moiety was unambiguously identified as a component of the fungal siderophore using mass spectroscopy. The protein structure shows that the substrate-binding pocket is defined by 17 amino acid residues, in contrast to both prokaryotic adenylation domains and to previous predictions based on modeling. Existing substrate prediction methods for NRPS adenylation domains fail for domains from eukaryotes due to the divergence of their signature sequences from those of prokaryotes. Thus, this new structure will provide a basis for improving prediction methods for eukaryotic NRPS enzymes which play important and diverse roles in the biology of fungi.

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  • Superantigens - powerful modifiers of the immune system

    Fraser, John; Arcus, VL; Kong, P; Baker, Edward; Proft, Thomas (2000)

    Journal article
    The University of Auckland Library

    Superantigens are powerful microbial toxins that activate the immune system by binding to class II major histocompatibility complex and T-cell receptor molecules. They cause a number of diseases characterized by fever and shock and are important virulence factors for two human commensal organisms, Staphylococcus aureus and Streptococcus pyogenes, as well as for some viruses. Their mode of action and variation around the common theme of over-stimulating T cells, provides a rich insight into the constant battle between microbes and the immune system.

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  • The Three-dimensional structure of a superantigen-like protein, SET3, from a pathogenicity island of the Staphylococcus aureus genome.

    Arcus, VL; Langley, Ries; Proft, Thomas; Fraser, John; Baker, Edward (2002-08-30)

    Journal article
    The University of Auckland Library

    The staphylococcal enterotoxin-like toxins (SETs) are a family of proteins encoded within the Staphylococcus aureus genome that were identified by their similarity to the well described bacterial superantigens. The first crystal structure of a member of the SET family, SET3, has been determined to 1.9 A (R = 0.205, R(free) = 0.240) and reveals a fold characteristic of the superantigen family but with significant differences. The SET proteins are secreted at varying levels by staphylococcal isolates, and seroconversion studies of normal individuals indicate that they are strongly antigenic to humans. Recombinant SETs do not exhibit any of the properties expected of superantigens such as major histocompatibility complex class II binding or broad T-cell activation, suggesting they have an entirely different function. The fact that the whole gene family is clustered within the pathogenicity island SaIn2 of the S. aureus genome suggests that they are involved in host/pathogen interactions.

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