title: Characterization of Staphylococcus aureus EssB, an integral membrane component of the Type VII secretion system: atomic resolution crystal structure of the cytoplasmic segment.
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.
authors.affiliation: College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.
authors.forename: Martin
authors.initials: M
authors.lastname: Zoltner
authors.forename: Paul K
authors.initials: PK
authors.lastname: Fyfe
authors.forename: Tracy
authors.initials: T
authors.lastname: Palmer
authors.forename: William N
authors.initials: WN
authors.lastname: Hunter
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D003593:Cytoplasm / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013211:Staphylococcus aureus / Q000235:genetics / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D020798:Two-Hybrid System Techniques
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins