Riviere, G.; Kumar, P.; Cummins, T.; Hsiao, A.; Mueller, L. J.

The New Delhi metallo- {beta} -lactamase 1 (NDM1) is a major driver of carbapenem resistance in Gram- negative pathogens, yet the molecular basis by which antimicrobial peptides inhibit this enzyme has remained unresolved. Thanatin, a disulfide- stabilized {beta} -hairpin peptide, was previously proposed to inactivate NDM1 by displacing catalytic Zn-ions, but this model lacked direct structural support. Here, we combine high-resolution NMR spectroscopy, intermolecular NOE mapping, HADDOCK-guided docking, and molecular dynamics simulations to reveal a distinct zinc-retaining dynamic allosteric mechanism. Thanatin binds adjacent to the catalytic groove, preserving the native di-zinc coordination environment while simultaneously rigidifying the L3 catalytic loop, as confirmed by Zn-bound spectral fingerprints and EDTA titration experiments. This conformational restriction explains how the peptide inhibits the enzyme while maintaining a zinc-bound but catalytically compromised state, a finding that contrasts with zinc-displacement hypotheses and reconciles prior biochemical observations with structural data. In bacterial assays, this allosteric inhibition translates to a moderate restoration of carbapenem sensitivity, resulting in a 50% reduction in viable cell output even under high-level enzyme expression. Our findings establish a mechanistic framework for designing next-generation peptide inhibitors that target the dynamic vulnerabilities of metallo- {beta} -lactamase.