Opening of a cryptic pocket in β-lactamase increases penicillinase activity

Significance A protein is a shape-shifter, but it is currently unclear which of the many structures a protein can adopt are relevant for its function. Here, we examine conformations that contain a “cryptic” pocket (i.e., a pocket absent in ligand-free structures). Cryptic pockets have potential utility in drug discovery efforts because they provide a means to target “undruggable” proteins (i.e., proteins lacking known pockets) or enhance rather than inhibit protein function. In this study, we use a combination of thiol-labeling and kinetic assays, NMR, and molecular dynamic simulations to identify the function of the Ω-loop cryptic pocket in β-lactamase enzymes. We find that an open pocket population is beneficial for hydrolysis of the substrate benzylpenicillin.