Peptidoglycan recruitment by a penicillin binding protein

The cell wall is essential for bacterial survival. Its core component is peptidoglycan (PG), a polymer comprised of disaccharide-peptides (stem peptides) that are cross-linked to one another via transpeptidation by penicillin-binding proteins (PBPs). While much is known about how PBPs are inactivated by β-lactam antibiotics, little is known about how PBPs bind and catalyze the transpeptidation of PG. Here we show how native PG and stem peptides are recruited to PBP5 of E. faecium, a critical ESKAPE pathogen. We discovered that PG binds PBP5 at the periphery of the PBP active site cleft, not the active site, and that the D-Ala leaving group contributes minimally to PBP binding. We show that β-lactam antibiotics and stem peptides can bind PBP5 simultaneously. We also show that only the single central residue of the stem peptide (L-Lys substituted by D-iAsn in E. faecium) is both necessary and sufficient for peptide recruitment. Finally, we translate our molecular findings by demonstrating that recruitment binding variants are unable to create a PG cell wall in E. faecium. Our studies define the key molecular interactions that govern bacterial cell wall formation and provide opportunities for the development of antibiotics that do not rely on PBP inactivation.