Structural and Dynamics Analyses of β-Lactam Inhibition of <i> <i>Streptococcus pneumoniae</i> </i> Penicillin-Binding Protein 1b (PBP1b) Guide Interrogation of Structure–Activity Relationships

The Gram-positive pathogen <i><i>Streptococcus pneumoniae</i></i>, like the majority of bacteria, contains a peptidoglycan-based cell wall whose structure is highly dependent on the action of penicillin-binding proteins (PBPs). While the β-lactam antibiotics have been employed as an antimicrobial strategy for nearly a century, much remains unclear about how inhibitor structure informs potency and PBP isoform selectivity. Here, we obtained high-resolution structures (<2Å) of <i><i>S. pneumoniae</i></i> PBP1b cocrystallized with 6 β-lactams. Surprisingly, 2 structures feature a noncanonical conformation of the covalent "acyl-enzyme complex." To clarify how protein-ligand interactions mediate inhibitor binding, we applied molecular modeling and molecular mechanics-based dynamics analyses. Our analyses illustrate how seemingly minimal changes to inhibitor structure modulate β-lactam binding mode and inhibitor potency, as described by the metric <i>k</i>_inact/<i>K</i>_I. Furthermore, we demonstrate that persistent interaction in the covalent acyl-enzyme complex between the inhibitor carboxylate and a highly conserved three-residue motif is not fully predictive of <i>k</i>_inact/<i>K</i>_I for PBP1b. <i>In silico</i> modeling suggests that the noncovalent preacyl complex may leverage this interaction, but a postacylation change in ligand conformation may accompany acylation in some inhibitors. The elucidation of key PBP1b ligand-receptor interactions pre- and postacylation will inform the rational design of novel PBP inhibitors and probes.