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    The role of chaperone-subunit usher domain interactions in the mechanism of bacterial pilus biogenesis revealed by ESI-MS

    Morrissey, B. and Leney, A.C. and Toste Rego, A. and Phan, G. and Allen, W.J. and Verger, D. and Waksman, Gabriel and Ashcroft, A.E. and Radford, S.E. (2012) The role of chaperone-subunit usher domain interactions in the mechanism of bacterial pilus biogenesis revealed by ESI-MS. Molecular & Cellular Proteomics 11 (7), ISSN 1535-9476.

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    The PapC usher is a β-barrel outer membrane protein essential for assembly and secretion of P pili that are required for adhesion of pathogenic E. coli, which cause the development of pyelonephritis. Multiple protein subunits form the P pilus, the highly specific assembly of which is coordinated by the usher. Despite a wealth of structural knowledge, how the usher catalyzes subunit polymerization and orchestrates a correct and functional order of subunit assembly remain unclear. Here, the ability of the soluble N-terminal (UsherN), C-terminal (UsherC2), and Plug (UsherP) domains of the usher to bind different chaperone-subunit (PapDPapX) complexes is investigated using noncovalent electrospray ionization mass spectrometry. The results reveal that each usher domain is able to bind all six PapDPapX complexes, consistent with an active role of all three usher domains in pilus biogenesis. Using collision induced dissociation, combined with competition binding experiments and dissection of the adhesin subunit, PapG, into separate pilin and adhesin domains, the results reveal why PapG has a uniquely high affinity for the usher, which is consistent with this subunit always being displayed at the pilus tip. In addition, we show how the different soluble usher domains cooperate to coordinate and control efficient pilus assembly at the usher platform. As well as providing new information about the protein-protein interactions that determine pilus biogenesis, the results highlight the power of noncovalent MS to interrogate biological mechanisms, especially in complex mixtures of species.


    Item Type: Article
    School: Birkbeck Faculties and Schools > Faculty of Science > School of Natural Sciences
    Research Centres and Institutes: Structural Molecular Biology, Institute of (ISMB)
    Depositing User: Sarah Hall
    Date Deposited: 20 Jan 2014 14:45
    Last Modified: 02 Aug 2023 17:09


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