Staphylococcus simulans biovar staphylolyticus lysostaphin efficiently cleaves Staphylococcus aureus cell walls. The protein is in late clinical trials as a topical anti-staphylococcal agent, and can be used to prevent staphylococcal growth on artificial surfaces. Moreover, the gene has been both stably engineered into and virally delivered to mice or livestock to obtain resistance against staphylococci. Here, we report the first crystal structure of mature lysostaphin and two structures of its isolated catalytic domain at 3.5, 1.78 and 1.26 Å resolution, respectively. The structure of the mature active enzyme confirms its expected organization into catalytic and cell-wall-targeting domains. It also indicates that the domains are mobile with respect to each other because of the presence of a highly flexible peptide linker. The high-resolution structures of the catalytic domain provide details of Zn2+ coordination and may serve as a starting point for the engineering of lysostaphin variants with improved biotechnological characteristics.Structured digital abstractlysostaphin by x-ray crystallography (1,2).

In a new, versatile approach to fun­ction­alizing recombinant spider silk, L‐azidohomoalanine is introduced residue‐specifically in the minispidroin protein 4RepCT through expression in an E. coli methionine auxotroph. Both fluorophores and the antibiotic levofloxacin are attached to this bio‐orthogonal amino acid using copper‐catalyzed click chemistry, either before or after the silk fibers are self‐assembled.

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.

Interaction of the TolB box of Group A colicins with the TolB protein in the periplasm of Escherichia coli cells promotes transport of the cytotoxic domain of the colicin across the cell envelope. The crystal structure of a complex between a 107-residue peptide (TA1–107) of the translocation domain of colicin A (ColA) and TolB identified the TolB box as a 12-residue peptide that folded into a distorted hairpin within a central canyon of the β-propeller domain of TolB. Comparison of this structure with that of the colicin E9 (ColE9) TolB box–TolB complex, together with site-directed mutagenesis of the ColA TolB box residues, revealed important differences in the interaction of the two TolB boxes with an overlapping binding site on TolB. Substitution of the TolB box residues of ColA with those of ColE9 conferred the ability to competitively recruit TolB from Pal but reduced the biological activity of the mutant ColA. This datum explains (i) the difference in binding affinities of ColA and ColE9 with TolB, and (ii) the inability of ColA, unlike ColE9, to competitively recruit TolB from Pal, allowing an understanding of how these two colicins interact in a different way with a common translocation portal in E. coli cells.

Lysostaphin (EC. 3.4.24.75) is a protein secreted by Staphylococcus simulans biovar staphylolyticus and has been shown to be active against methicillin resistant S. aureus (MRSA). The design and synthesis of three internally quenched substrates for lysostaphin based on the peptidoglycan crossbridges of S. aureus, and their use in fluorescence resonance energy transfer (FRET) assays is reported. These substrates enabled the gathering of information about the endopeptidase activity of lysostaphin and the effect that mutations have on its enzymatic ability. Significant problems with the inner filter effect and substrate aggregation were encountered; their minimisation and the subsequent estimation of the kinetic parameters for the interaction of lysostaphin with the substrates is described, as well as a comparison of substrates incorporating two FRET pairs: Abz-EDDnp and DABCYL-EDANS. In addition to this, the points of cleavage caused by lysostaphin in Abz-pentaglycine-EDDnp have been determined by HPLC and mass spectrometry analysis to be between glycines 2 and 3(approximately 60%) and glycines 3 and 4 (approximately 40%).