Structural bases of peptidoglycan recognition by lysostaphin SH3b domain

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.

Figure 1

(A) Structural comparison of the lysostaphin SH3b domain in complex with the pentaglycine peptide (dark green; PDB 5LEO) with the SH3b domains of Ale-1 (gray; PDB 1R77) and the apo-lysostaphin (brown; PDB 4LXC). (B) Alignment of amino acid sequence of SH3b domains from lysostaphin and Ale-1. Residues involved in interactions with pentaglycine are marked as red.

Figure 2

Cartoon representation and topology of the lysostaphin SH3b (green) and eukaryotic SH3 domains (blue).

Figure 3

Structure of lysostaphin SH3b domain in complex with pentaglycine. (A) front view, (B) Close up view of residues interacting with the pentaglycine peptide, (C) Calculated composite omit electron density map of pentaglycine at 1 σ.

Figure 4

Peptide binding sites on the surface of lysostaphin SH3b domain predicted using DoGSiteScorer webserver.

Figure 5

(A) Structure of ligand (PG fragment) used for modeling. Background colors (cyan for the cross-linked stem peptide, yellow for the pentapeptide bridge, orange for the bridge-linked stem peptide) corresponds to colors of the ligands’ main chain in the panels (B–E). Dashed arrows indicate directions of the peptidoglycan network. The terminal amino groups of the fragments used for modeling were charged, however in the muramoyl-linked stem peptide in the peptidoglycans, these groups are neutral (B–E). Predictions of the binding modes of PG-ligands with SH3b domain of lysostaphin: PG-fragment (B–D) and PG-G3S fragment (E). Poses B and E had been initially obtained using molecular docking and then confirmed as stable conformations by Molecular Dynamics simulations. Poses C and D have been obtained and confirmed as stable during Molecular Dynamics exploration of the conformational space of the PG-fragment upon binding SH3b.

Figure 6

Fluorimetric binding assay of wild type LssSH3b_GFP and its variants (N405A, Y472A, N405/Y472) with various strains of S. aureus which represent wild type (pentaglycine) or altered cross-bridges. Results were normalized to the % of binding of wild type LssSH3b_GFP domain to wild type S. aureus NCTC 8325-4 (100%). The error bars represent standard deviation calculated for three experiments each run with triplicates.

Figure 7

Lytic activity of lysostaphin with substituted residues in SH3b domain. (A) 10 nM enzymes activity was measured within 2 h at room temperature, (B) 100 nM enzymes activity was measured within 1 h at room temperature. Results present the percentage of reduction of initial OD₆₀₀ (mean value from 3 independent experiments in triplicates, error bars represent standard deviation).

Figure 8

Binding of mature lysostaphin to pentaglycine. Two molecules in the asymmetric unit of the mature lysostahin (PDB:4LXC) were superimposed to show the flexibility of the domains. The catalytic domains (yellow and gray) differ in orientation by almost 100°. Catalytic zinc ion shown as a ball and SH3b domains are green. Moreover, the structure of mature lysostaphin was superimposed with ligands from complex structures of LytM catalytic domain with substrate analogue (PDB:4ZYB) and LssSH3b domain with pentaglycine (PDB:5LEO).