Inhibitor-Dependent Tolerance of New Delhi Metallo-β-Lactamase Driven by Single Mutation-Induced Conformational Changes

The emergence of metallo-β-lactamases as formidable adversaries in the antimicrobial resistance crisis stems from their unparalleled capacity to hydrolyze β-lactam antibiotics. This study deciphers the evolutionary strategy of New Delhi metallo-β-lactamase (NDM) variants through studies of conformational dynamics. We employ hydrogen/deuterium exchange mass spectrometry (HDX-MS) to map conformational landscapes of NDM in the ligand-free state and in the bound states with inhibitors l-captopril, d-captopril, ebselen, and aspergillomarasmine A (AMA), respectively. Crucially, our findings reveal similar allosteric fingerprints corresponding to different inhibition mechanisms; that is, inhibition induces pronounced dynamic perturbations in the α3-L8-β8 region─a previously under-characterized region. Strikingly, the clinically prevalent M154L mutation in this region reshapes conformational flexibility, amplifying inhibitor-specific conformational responses without altering the l/d-captopril binding dynamics. This study demonstrates how a single mutation can be critical for antibiotic resistance evolution where zinc is scarce in the presence of AMA and ebselen, as indicated by more protected HDX patterns of the α3-L8 region and several active-site loop (ASL) regions. Our results establish three key advances: (1) identification of α3-L8 as a cryptic allosteric region governing conformational adaptability, (2) demonstration of a single mutation M154L rewiring long-range dynamic communication, and (3) proposal of conformation-guided inhibitor design as a viable strategy against NDM. Overall, this work unveils a novel perspective─resistance mutations function not merely as chemical optimizers but as allosteric modulators that exploit inherent protein plasticity. These insights position the α3-L8 region as a compelling target for developing novel inhibitors, providing a blueprint for combating the next frontier of antimicrobial resistance.