Allosteric Rewiring in NDM-4: Protein Energy Network Analysis Reveals Mechanical Decoupling of the Catalytic Zinc and L3 Canopy Loop via the M154L Substitution

The New Delhi metallo-β-lactamase-1 (NDM-1) is a primary driver of global carbapenem resistance. Its clinical variant, NDM-4,characterized by a single M154L substitution, exhibits significantly enhanced catalytic efficiency. Because this mutation is locatedoutside the primary coordination sphere, its enhanced activity must rely on an unmapped allosteric mechanism. To decode thisstructural rewiring, we applied a Protein Energy Network (PEN) framework utilizing an Anisotropic Network Model (ANM) tocompare the high-resolution apo-states of NDM-1 and NDM-4. By calculating shifts in Eigenvector Centrality (ΔZ) across theharmonic spring networks, we identified a long-range mechanical "see-saw" effect induced by the rigidifying M154L substitution.Our topological mapping demonstrates that the mutation transfers mechanical stress into the distal L10 loop (residues 213–224,max ΔZ = +1.229), which acts as an allosteric stress sink. Concurrently, this shift drains mechanical rigidity from the active siteflank, the L3 canopy loop (residues 68–71, min ΔZ = -1.320), and the primary catalytic zinc ion (ZN302, ΔZ = -0.913). Thisdecoupling isolates the active site from the rigid core of the enzyme, increasing localized dynamic flexibility. We conclude that thisspecific allosteric rewiring grants NDM-4 the structural flexibility necessary to accommodate and hydrolyze bulkier carbapenemsubstrates, identifying the L10 loop as a potential target for novel allosteric inhibitors.