Molecular Insights into the β‑Lactam α‑Methyl Modulation of Pseudomonas aeruginosa Penicillin-Binding Protein 3...

The stereochemical recognition of the α-methyl group at the d-Ala-d-Ala terminus of peptidoglycan by penicillin-binding proteins (PBPs) has been implicated in shaping the evolutionary divergence of dd-peptidases. Here, we investigate how the β-lactam α-substituent identity influences the conformational dynamics of wild-type Pseudomonas aeruginosa PBP3 using molecular dynamics simulations of covalent acyl-enzyme complexes with CEF_acyl (α-hydro) and its α-methyl derivative, MEC_acyl. Our analysis reveals that the α-methyl group in MEC-PBP3_acyl is accommodated within a defined methyl pocket predominantly composed of conserved residues K297, S349, N351, and V333. Hydration network into the buried active site is disrupted in the MEC-PBP3_acyl complex, which consequently results to the loss of a deacylation-competent geometry of water at K297 toward the β-lactam acyl carbon required for hydrolytic deactivation. Time-resolved analyses of the CEF-PBP3_acyl simulation reveals that the contraction of the active site α-loop is associated with the coupling of water bridge networks that connects the α2 helix active site motif ₂₉₄ STVK ₂₉₇ to a distal salt bridge cluster-the "water sink" (R284 _α1b, D288 _loop, R504 _β4, and D525 _β5) which corroborates crystal structure evidence (PDB ID: 6R3X) [BelliniD., J. Mol. Biol.2019, 431, 3501-3519]. Pocket-based analyses show that the expansion of the methyl pocket into the STVK motif coincides with active site solvation. These dynamic observations are observed to be associated with the shifting salt bridge interactions of R504 _β4 on the α-loop, which may rationalize resistance in R504 mutants. The steric bulk of the α-methyl group in MEC_acyl toward the K297 _α2 side chain disables active site plasticity and consequently impairs the loop mobility required for the influx of water into the active site. These findings provide mechanistic molecular insights into how α-substituent chemistry modulates active site hydration dynamics in PBP3 and support the importance of α-methyl recognition in dd-peptidases. This work also establishes a structural framework for future studies of PBPs and β-lactam drug design relevant to antimicrobial resistance.