Characterization of Staphylococcus aureus EssB, an integral membrane component of the Type VII secretion system: atomic resolution crystal structure of the cytoplasmic segment

Martin Zoltner, Paul K. Fyfe, Tracy Palmer, William N. Hunter

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    14 Citations (Scopus)

    Abstract

    The Type VII protein translocation/secretion system, unique to Gram-positive bacteria, is a key virulence determinant in Staphylococcus aureus. We aim to characterize the architecture of this secretion machinery and now describe the present study of S. aureus EssB, a 52 kDa bitopic membrane protein essential for secretion of the ESAT-6 (early secretory antigenic target of 6 kDa) family of proteins, the prototypic substrate of Type VII secretion. Full-length EssB was heterologously expressed in Escherichia coli, solubilized from the bacterial membrane, purified to homogeneity and shown to be dimeric. A C-terminal truncation, EssB?C, and two soluble fragments termed EssB-N and EssB-C, predicted to occur on either side of the cytoplasmic membrane, have been successfully purified in a recombinant form, characterized and, together with the full-length protein, used in crystallization trials. EssB-N, the 25 kDa N-terminal cytoplasmic fragment, gave well-ordered crystals and we report the structure, determined by SAD (single-wavelength anomalous diffraction) targeting an SeMet (selenomethionine) derivative, refined to atomic (1.05 Å; 1 Å = 0.1 nm) resolution. EssB-N is dimeric in solution, but crystallizes as a monomer and displays a fold comprised of two globular domains separated by a cleft. The structure is related to that of serine/threonine protein kinases and the present study identifies that the Type VII secretion system exploits and re-uses a stable modular entity and fold that has evolved to participate in protein-protein interactions in a similar fashion to the catalytically inert pseudokinases.
    Original languageEnglish
    Pages (from-to)469-477
    Number of pages9
    JournalBiochemical Journal
    Volume449
    Issue number2
    DOIs
    Publication statusPublished - 15 Jan 2013

    Keywords

    • early secretory antigenic target of 6 kDa system 1 (ESX-1)
    • Gram-positive bacterium
    • protein kinase
    • PROTEIN SECRETION
    • pseudokinase
    • X-ray crystallography

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