title: Expression, purification and crystallization of a membrane-associated, catalytically active type I signal peptidase from Staphylococcus aureus.
abstract: Staphylococcus aureus infections are becoming increasingly difficult to treat as they rapidly develop resistance to existing antibiotics. Bacterial type I signal peptidases are membrane-associated, cell-surface serine proteases with a unique catalytic mechanism that differs from that of eukaryotic endoplasmic reticulum signal peptidases. They are thus potential antimicrobial targets. S. aureus has a catalytically active type I signal peptidase, SpsB, that is essential for cell viability. To elucidate its structure, the spsB gene from S. aureus Newman strain was cloned and overexpressed in Escherichia coli. After exploring many different protein-modification constructs, SpsB was expressed as a fusion protein with maltose-binding protein and crystallized by hanging-drop vapour diffusion. The crystals belonged to the monoclinic space group P2(1) and diffracted to 2.05 Å resolution. The crystal structure of SpsB is anticipated to provide structural insight into Gram-positive signal peptidases and to aid in the development of antibacterial agents that target type I signal peptidases.
authors.affiliation: School of Biological Sciences, University of Auckland, Auckland 1010, New Zealand.
authors.forename: Yi Tian
authors.initials: YT
authors.lastname: Ting
authors.affiliation: School of Biological Sciences, University of Auckland, Auckland 1010, New Zealand.
authors.forename: Gaëlle
authors.initials: G
authors.lastname: Batot
authors.affiliation: School of Biological Sciences, University of Auckland, Auckland 1010, New Zealand.
authors.forename: Edward N
authors.initials: EN
authors.lastname: Baker
authors.affiliation: School of Biological Sciences, University of Auckland, Auckland 1010, New Zealand.
authors.forename: Paul G
authors.initials: PG
authors.lastname: Young
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D003460:Crystallization; D018360:Crystallography, X-Ray; D058619:Maltose-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D013211:Staphylococcus aureus / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; C026515:type I signal peptidase
keywords: SpsB; Staphylococcus aureus; cell secretion; maltose-binding fusion protein; type I signal peptidase