Allosteric inhibition of NDM-1 by thanatin preserves the di-zinc center while restricting dynamics

The New Delhi metallo-β-lactamase 1 (NDM-1) 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 β-hairpin peptide, was previously proposed to inactivate NDM-1 by displacing catalytic Zn²⁺ 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-β-lactamases.