Discovery of a Cryptic Pocket in EcDsbA Opens New Opportunities for Antibacterial Discovery

We have used nuclear magnetic resonance (NMR) spectroscopy to characterize dynamics in the bacterial oxidoreductase enzyme Escherichia coli disulfide bond protein A (EcDsbA). Through this process we identified a cryptic pocket in the structure. We demonstrate that we can identify small molecule "fragments" that bind entirely within this cryptic site. The fragments bind to the cryptic pocket with unusually slow kinetics and a preference for interacting with the oxidized state of EcDsbA where the two cysteine residues at the active site form a disulfide bond. We characterize the mechanism of binding, involving conformational changes in the active-site helix of EcDsbA, which are observed preferentially in the oxidized state. This dynamics-driven binding mechanism explains both the slow kinetics and the redox-dependent binding of the ligands. Furthermore, we demonstrate that compounds binding to the cryptic pocket inhibit EcDsbA activity. These findings highlight the value of dynamics data in identification of the cryptic pocket and identify a new target site for developing more potent inhibitors of EcDsbA.