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.