Functional and structural basis of peptidoglycan recognition by the SH3_5 domain family

A large number of cell surface proteins contain an SH3 type 5 domain (SH3_5) responsible for the cell wall binding of these proteins to peptidoglycan. SH3_5 domains are involved in the recognition of peptidoglycan in different pathogenic bacteria including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VRE). This work sought to elucidate the mechanisms underpinning the recognition of peptidoglycan by SH3_5 proteins. As a model system, we focused on the C-terminal SH3_5 domain from the lysostaphin enzyme. Lysostaphin is a bacteriolytic enzyme produced by S. simulans that cleaves the pentaglycine crossbridge in S. aureus peptidoglycan. This potent enzyme can efficiently lyse MRSA strains and disrupt biofilms. The lysostaphin enzyme contains an N-terminal catalytic domain with glycylglycil endopeptidase activity and a C-terminal SH3_5 cell wall targeting domain. Nuclear Magnetic Resonance (NMR) titrations, X ray crystallography, and mutational analyses, revealed how this SH3_5 domain efficiently recognises S. aureus peptidoglycan. Our NMR titrations showed that the pentaglycine crossbridge and the peptide stem are recognized by two independent binding sites located on opposite sides of the SH3_5 domain. The 1.4 Å crystal structure of the SH3_5 domain, in complex with a synthetic peptidoglycan fragment, revealed a dual SH3_5 binding mechanism whereby the G5 ligand from a monomeric fragment was recognized by one SH3_5 domain and the P4 peptide was bound to another SH3_5 protein. We propose that this binding mechanism leads to a synergistic and structurally dynamic binding, which induces clustering of the SH3_5 domains and increases the local concentration of the enzyme resulting in its potent bacteriolytic activity. We propose that this research will serve as a framework for understanding how bacteria control the enzymatic activity of PG hydrolases and will contribute to the development of antimicrobial approaches.