Thiol Labeling Reveals Presence of Cryptic Binding Sites in β-Lactamase

Abstract

The field of drug design has for many years sought to design ligands for known binding pockets in proteins. Cryptic binding sites, which are unexpected openings that are invisible to normal experimental methods but can modulate activity through communication with the active site, have the ability to greatly expand the number of proteins whose behavior and activity can be easily modulated by increasing the number of druggable sites. Recent work has offered the possibility of identifying these sites from molecular dynamics simulations of proteins in the absence of ligands, allowing identification of sites for many ligands to be screened in a single experiment. To experimentally verify these computational models, we have tested β-lactamase for the presence of cryptic binding sites. We have employed a method using mutagenesis and thiol labeling to verify the transient opening of cryptic sites as well as the ability of a large, covalent adduct at these sites to alter activity of the protein. This has led us to conclude that conformations with transiently open pockets exist in equilibrium with the known structure under native conditions, and these pockets may be suitable for future drug design studies.