Abstract
Staphylococcus simulans lysostaphin cleaves pentaglycine cross-bridges between stem peptides in the peptidoglycan of susceptible staphylococci, including S. aureus. This enzyme consists of an N-terminal catalytic domain and a cell wall binding domain (SH3b), which anchors the protein to peptidoglycan. Although structures of SH3bs from lysostaphin are available, the binding modes of peptidoglycan to these domains are still unclear. We have solved the crystal structure of the lysostaphin SH3b domain in complex with a pentaglycine peptide representing the peptidoglycan cross-bridge. The structure identifies a groove between β1 and β2 strands as the pentaglycine binding site. The structure suggests that pentaglycine specificity of the SH3b arises partially directly by steric exclusion of Cβ atoms in the ligand and partially indirectly due to the selection of main chain conformations that are easily accessible for glycine, but not other amino acid residues. We have revealed further interactions of SH3b with the stem peptides with the support of bioinformatics tools. Based on the structural data we have attempted engineering of the domain specificity and have investigated the relevance of the introduced substitutions on the domain binding and specificity, also in the contexts of the mature lysostaphin and of its bacteriolytic activity.
Highlights
Most peptidoglycan hydrolases, those of phage origin, have modular structure and are built of at least one catalytic and one cell wall binding domain (CBD)[1]
In our previous work we have demonstrated that addition of lysostaphin SH3b domain to the catalytic domain of LytM, an autolysin from S. aureus, made the enzyme more salt-tolerant[17]
As reported already for Ale-1, the prokaryotic SH3b domains are built of eight β-strands (β1–β8) that can roughly be divided into two sheets packed at the right angle against each other
Summary
Those of phage origin, have modular structure and are built of at least one catalytic and one cell wall binding domain (CBD)[1]. Different CBDs recognize and bind noncovalently to various elements of the cell walls, like the peptidoglycan sugar backbone (LysM)[6], choline (Cpl-1)[3], teichoic acids (R domain)[7], and peptidoglycan cross-bridges (SH3b)[8]. SH3b domains that are present in many staphylococcal phage endolysins and some autolysins, are well conserved[11,12] These domains are necessary for accurate cell wall recognition[8,13,14] and have been shown to recognize and bind glycine cross-bridges characteristic for most staphylococci[15]. We show that these interactions are crucial for the function of lysostaphin and we attempt structure based engineering of the domain specificity
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