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.