Cryptic Binding Pockets in PDC‑3 β‑Lactamase Modulate Resistance Profiles.

Cryptic binding pockets in proteins can modulate catalysis, allostery, and druggability. Yet they are rarely captured by experiments or conventional molecular dynamics simulations. Here, we combine enhanced sampling with an unsupervised deep-learning pipeline to map the full conformational landscape of the Ω-loop in class C β-lactamase PDC-3. Three principal conformational ensembles were identified: a crystal-like state resembling the native structure, an expansive state characterized by widening of the active-site cleft, and a constricted state that blocks access to the catalytic site. Residues 219 and 221 act as molecular switches that shuffle the enzyme between these states and thereby modulate the resistance profiles. Steady-state inhibition assays with nitrocefin and bulky cephalosporins confirm that substitutions at these positions selectively reshaped the binding pocket. In addition, across multiple expansive states, D217 repeatedly forms a salt bridge with K67 in a geometry reminiscent of general base E166 of class A enzymes. Thus, it is plausible that D217 might transiently adopt a 'backup' general base role under certain conformational states. Most strikingly, occlusion of the catalytic site reveals a previously unseen cryptic pocket, offering an attractive allosteric target for inhibitors that would lock PDC-3 in a catalytically incompetent conformation. The integrated framework proposed in this study is highly generalizable and can serve as a powerful tool for identifying hidden protein conformations and uncovering previously inaccessible regulatory mechanisms.