Two-site recognition of Staphylococcus aureus peptidoglycan by lysostaphin SH3b

Abstract

Lysostaphin is a bacteriolytic enzyme targeting peptidoglycan, the essential component of the bacterial cell envelope. It displays a very potent and specific activity toward staphylococci, including methicillin-resistant Staphylococcus aureus. Lysostaphin causes rapid cell lysis and disrupts biofilms, and is therefore a therapeutic agent of choice to eradicate staphylococcal infections. The C-terminal SH3b domain of lysostaphin recognizes peptidoglycans containing a pentaglycine crossbridge and has been proposed to drive the preferential digestion of staphylococcal cell walls. Here we elucidate the molecular mechanism underpinning recognition of staphylococcal peptidoglycan by the lysostaphin SH3b domain. We show that the pentaglycine crossbridge and the peptide stem are recognized by two independent binding sites located on opposite sides of the SH3b domain, thereby inducing a clustering of SH3b domains. We propose that this unusual binding mechanism allows synergistic and structurally dynamic recognition of S. aureus peptidoglycan and underpins the potent bacteriolytic activity of this enzyme.

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Fig. 1: Mapping the interaction surface of the SH3b domain with synthetic S. aureus PG fragments.
Fig. 2: Structure of lysostaphin SH3b in complex with the P4–G5 ligand.
Fig. 3: Binding activity of recombinant SH3b–mNeonGreen proteins to purified S. aureus PG.
Fig. 4: Models for binding of a ligand at two sites.

Data availability

Structural data have been deposited in the PDB with coordinate accession numbers 6RK4 (high-resolution set) and 6RJE (home source set). All other data generated or analyzed during this study are included in this article and its supplementary information or are available from the corresponding authors upon request.

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Acknowledgements

L.S.G.-D. is a PhD student funded by the Mexican government through a CONACYT scholarship. H.W.-M. is supported by a BBSRC MIBTP studentship. We thank the BBSRC and EPSRC for funding to upgrade the 600- and 800-MHz spectrometers, respectively (grant numbers BB/R000727/1 and EP/S01358X/1). The work in the laboratory of I.S. is supported by the Foundation for Polish Science (FNP) program, co-financed by the European Union under the European Regional Development Fund (grant TEAMTECH/2016-3/19).

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S.M. conceived the project and designed experiments with M.P.W. and A.L.L. A.M.H. and A.J.R. assigned the SH3b spectrum. L.S.G.-D. carried out all NMR experiments and analyzed them with the help of A.M.H. and M.P.W. B.S. and L.S.G.-D. built all SH3b recombinant proteins to carry out functional assays and crystallographic analyses. A.W.-M. crystallized the protein and solved the structure with the help of A.L.L. E.J. and I.S. provided reagents. L.S.G.-D., H.W.-M., B.S., A.M.H., M.P.W., A.L.L. and S.M. analyzed the data. L.S.G.-D., S.M., A.L.L. and M.P.W. wrote the manuscript.

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Correspondence to Mike P. Williamson or Andrew L. Lovering or Stéphane Mesnage.

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Gonzalez-Delgado, L.S., Walters-Morgan, H., Salamaga, B. et al. Two-site recognition of Staphylococcus aureus peptidoglycan by lysostaphin SH3b. Nat Chem Biol 16, 24–30 (2020). https://doi.org/10.1038/s41589-019-0393-4

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