title: Design of a polypeptide FRET substrate that facilitates study of the antimicrobial protease lysostaphin.
abstract: We have developed a polypeptide lysostaphin FRET (fluorescence resonance energy transfer) substrate (MV11F) for the endopeptidase activity of lysostaphin. Site-directed mutants of lysostaphin that abolished the killing activity against Staphylococcus aureus also completely inhibited the endopeptidase activity against the MV11 FRET substrate. Lysostaphin-producing staphylococci are resistant to killing by lysostaphin through incorporation of serine residues at positions 3 and 5 of the pentaglycine cross-bridge in their cell walls. The MV11 FRET substrate was engineered to introduce a serine residue at each of four positions of the pentaglycine target site and it was found that only a serine residue at position 3 completely inhibited cleavage. The introduction of random, natural amino acid substitutions at position 3 of the pentaglycine target site demonstrated that only a glycine residue at this position was compatible with lysostaphin cleavage of the MV11 FRET substrate. A second series of polypeptide substrates (decoys) was developed with the GFP (green fluorescent protein) domain of MV11 replaced with that of the DNase domain of colicin E9. Using a competition FRET assay, the lysostaphin endopeptidase was shown to bind to a decoy peptide containing a GGSGG cleavage site. The MV11 substrate provides a valuable system to facilitate structure/function studies of the endopeptidase activity of lysostaphin and its orthologues.
authors.affiliation: Institute of Infection, Immunity and Inflammation, School of Molecular Medical Sciences, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham, U.K.
authors.forename: Philip
authors.initials: P
authors.lastname: Bardelang
authors.forename: Mireille
authors.initials: M
authors.lastname: Vankemmelbeke
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhang
authors.forename: Hannah
authors.initials: H
authors.lastname: Jarvis
authors.forename: Eleni
authors.initials: E
authors.lastname: Antoniadou
authors.forename: Sophie
authors.initials: S
authors.lastname: Rochette
authors.forename: Neil R
authors.initials: NR
authors.lastname: Thomas
authors.forename: Christopher N
authors.initials: CN
authors.lastname: Penfold
authors.forename: Richard
authors.initials: R
authors.lastname: James
mesh_terms: D003001:Cloning, Molecular; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D008248:Lysostaphin / Q000737:chemistry* / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*; D013211:Staphylococcus aureus / Q000187:drug effects
chemical_list: D010455:Peptides; D010450:Endopeptidases; C021524:lysostaphin endopeptidase; D008248:Lysostaphin