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.

Crystal structure of the antimicrobial peptidase lysostaphin from Staphylococcus simulans

“…The expression plasmid for lysostaphin (pET21) encoded the catalytic and CWT domains (residues 248–493, UniProt entry P10547) and a C-terminal His-tag (LEHHHHHH sequence) under the control of the T7 promoter and lac repressor, as described previously 44. BL21(DE3) Escherichia coli cells were grown in 2× YT media to a D 600 of 0.6, induced with 1.2 m m isopropyl β- d -thiogalactoside, and grown for a further 3 h at 30 °C.…”

Bacteriophage-derived CHAP domain protein, P128, kills Staphylococcus cells by cleaving interpeptide cross-bridge of peptidoglycan

“…A number of Staphylococcus species are known to be naturally resistant to lysostaphin because of the presence of Fem-like proteins, which incorporate serine in place of a glycine in the pentaglycine cross-bridge of the peptidoglycan (DeHart et al, 1995;Sugai et al, 1997;Ehlert et al, 2000). In a study involving FRET (fluorescence resonance energy transfer) assays, replacing the third glycine of a GGGGG sequence with a serine was shown to abolish the activity of lysostaphin on the FRET substrate (Bardelang et al, 2009). Apart from those in fem genes, mutations in other genes have also been reported to confer resistance to lysostaphin.…”

Gene and Protein Sequence Optimization for High-Level Production of Fully Active and Aglycosylated Lysostaphin in Pichia pastoris

“…In the current study of LST glycosylation, all mutations at the Nlinked N125 residue led to significantly decreased enzyme activity, whereas mutations at two adjacent residues (S126 and T127) yielded fully functional aglycosylated variants. The severely compromised activity of the N125 mutants was especially surprising given the fact that residue 125 is thought to be located at the C-terminal end of the catalytic domain, and prior studies of key LST catalytic residues did not identify N125 (2). Molecular modeling of the LST catalytic domain suggested that substitutions at position 125 caused minimal structural perturbation and affected the peptide backbone even less than mutations at positions 126 and 127 (data not shown).…”

“…LST is synthesized as a preproenzyme of 493 amino acids, and its pre-(36 amino acids) and pro-(211 amino acids) sequences are removed during and after secretion, respectively. Mature LST is a monomer composed of an N-terminal catalytic domain (132 amino acids), a C-terminal cell wall binding domain (102 amino acids), and a short connecting linker (13 amino acids) between the two (2). The LST enzyme selectively and efficiently degrades pentaglycine cross-links in the peptidoglycan component of S. aureus cell walls, ultimately resulting in bacterial lysis and death.…”