Our work arose from the collaboration between three research groups, led by Stéphane Mesnage and Mike P. Williamson at the University of Sheffield (UK) and Andrew L. Lovering at the University of Birmingham (UK). The Mesnage lab studies the bacterial cell wall of Gram-positive pathogens, including the opportunistic nosocomial Staphylococcus aureus, with a main research focus on protein-cell wall interactions. The Williamson lab is using NMR to explore protein structural dynamics and macromolecular interactions in solution. The Lovering lab has expertise in X-ray crystallography.
Lysostaphin is a bacteriolytic enzyme that has attracted a lot of attention since its discovery in 1964 (1), due to its potent activity against staphylococci, notably methicillin-resistant S. aureus (2-4). Several studies have shown that lysostaphin can be used as a therapeutic agent to combat infections. We sought to investigate how the cell wall targeting domain (SH3b) of lysostaphin specifically targets S. aureus peptidoglycan (PG), the essential component of the bacterial cell wall.
Our work revealed a novel binding mechanism underpinned by a two-site recognition of S. aureus PG by the lysostaphin SH3b domain. We initially performed a series of NMR titrations with a set of ligands of increasing complexity and to our surprise, we found that the SH3b domain recognised not only the pentaglycine bridges but also the tetrapeptide stems via a different set of residues located at opposite sides of the protein surface. Intriguingly, the SH3b domain exhibited equally weak binding affinities to either of these two peptides moieties.
A major challenge was to understand these experimental results, given that the two recognition sites are on opposite sides of the protein, too far apart for the branched peptide stem to bind both sites simultaneously. We therefore decided to characterize the SH3b peptidoglycan interaction using X-ray crystallography. Co-crystallisation experiments allowed us to solve the high resolution (1.4 Å) crystal structure of the SH3b domain in complex with a synthetic branched peptide (Figure 1), made of a tetrapeptide stem substituted by a pentaglycine chain. This structure sheds light on the mechanism underpinning peptidoglycan recognition by lysostaphin. It revealed an unusual binding mechanism whereby the pentaglycine stem is recognised by one SH3b domain while the tetrapeptide stem is recognised by a distinct SH3b domain. This led us to propose a model explaining how this dual (and relatively low affinity) binding mechanism allows the enzyme to exhibit extremely potent activity against S. aureus PG. By having two binding sites on the same domain but arranged such that they are mutually exclusive, the protein can move across the PG surface without detaching from the substrate, maximizing the probability to have at least one site bound to the substrate at any time. We therefore proposed that the weak binding affinities measured by NMR for two distinct sites on the peptidoglycan molecule could allow the lysostaphin to “walk” along the PG surface to ensure the processivity of the catalytic domain. We also observed SH3b clustering in the presence of complex PG fragments, thereby increasing the effective concentration of lysostaphin at the PG surface. This phenomenon could explain why this enzyme is “punching” holes in the cell wall as seen by atomic force microscopy (5).
By Luz S. Gonzalez-Delgado and Hannah Walters-Morgan
Figure 1. Context of ligand-binding in relation to full-length lysostaphin. Our dual SH3b domains (light and dark blue) bound to ligand (yellow, stick form) were used as a basis to superpose the full-length structure of lysostaphin (two monomers taken from PDB 4LXC, coloured tan, active site and Zn ion in stick form and sphere, respectively). In this conformation, lysostaphin activity would act quite distal to the SH3 ligand-binding interface
Our paper: Gonzalez-Delgado, L.S., Walters-Morgan, H., Salamaga, B. et al. Two-site recognition of Staphylococcus aureus peptidoglycan by lysostaphin SH3b. Nat Chem Biol (2019) doi:10.1038/s41589-019-0393-4
References: 1. Schindler CA, & Schuhardt, V. T. Lysostaphin: a new bacteriolytic agent for the Staphylococcus. Proc Natl Acad Sci USA. 1964;51(3):414–21. 2. Climo MW, Patron, R. L., Goldstein, B. P., & Archer, G. L. . Lysostaphin treatment of experimental methicillin-resistant Staphylococcus aureus aortic valve endocarditis. Antimicrobial agents and chemotherapy. 1998;42(6):1355–60. 3. Kokai-Kun J. F. CT, Mond J.J. Lysostaphin as a treatment for systemic Staphylococcus aureus infection in a mouse model Journal of Antimicrobial Chemotherapy. 2007;60(5):1051-9. 4. Satishkumar R, Sankar, S., Yurko, Y., Lincourt, A., Shipp, J., Heniford, B. T., & Vertegel, A. Evaluation of the antimicrobial activity of lysostaphin-coated hernia repair meshes. Antimicrobial agents and chemotherapy. 2011;55(9):4379–85. 5. Francius G, Domenech, O., Mingeot-Leclercq, M. P., & Dufrêne, Y. F. Direct Observation of Staphylococcus aureus Cell Wall Digestion by Lysostaphin. Journal of bacteriology. 2008;190(24):7904–9.