Defining an allosteric circuit in the cysteine protease domain of Clostridium difficile toxins

Abstract

An internal cysteine protease domain (CPD) autoproteolytically regulates Clostridium difficile glucosylating toxins by releasing a cytotoxic effector domain into target cells. CPD activity is itself allosterically regulated by the eukaryotic-specific molecule inositol hexakisphosphate (InsP ₆). Although allostery controls the function of most proteins, the molecular details underlying this regulatory mechanism are often difficult to characterize. Here we use chemical probes to show that apo-CPD is in dynamic equilibrium between active and inactive states. InsP ₆ dramatically shifts this equilibrium towards an active conformer that is further restrained upon binding a suicide substrate. Structural analyses combined with systematic mutational and disulfide bond engineering studies reveal that residues within a β-hairpin region functionally couple the InsP ₆ binding site to the active site. Collectively, our results identify an allosteric circuit that allows bacterial virulence factors to sense and respond to the eukaryotic environment.