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.