Ligand binding into a cryptic pocket in Escherichia coli DsbA inhibits the enzymatic activity in vitro

Disulfide bond protein A (DsbA) plays a pivotal role in catalysing the formation of disulfide bonds within the periplasm of most Gram-negative bacteria. As this process is required for the folding of multiple virulence-associated proteins, inhibitors of DsbA have the potential to be developed as novel anti-virulence agents. Despite extensive efforts to develop high-affinity inhibitors targeting the canonical DsbA substrate-binding site, success has been limited. In the current work, we describe a dynamics-driven fragment discovery approach. Our analysis of EcDsbA dynamics using nuclear magnetic resonance relaxation-dispersion data revealed a novel “cryptic” ligand binding pocket within Escherichia coli DsbA (EcDsbA). Compounds bind to this cryptic pocket with unusually slow kinetics and a preference for interacting with oxidised EcDsbA, the predominant form of the protein found in the periplasm. We characterise an induced-fit mechanism, involving conformational changes in the active-site helix of EcDsbA, observed preferentially in the oxidised state, which facilitates access to the cryptic pocket. This conformational exchange 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 in vitro. These findings highlight the potential of the cryptic pocket as a target site for developing more potent inhibitors of EcDsbA.