Current treatment options for Clostridioides difficile infections of the gut are limited by reliance on antibiotics, resulting in high recurrence and mortality. Since the secreted toxin TcdB is the source of pathogenesis, a more effective treatment route may target TcdB directly. Allosteric control of the cysteine protease domain (CPD) of TcdB offers a promising intervention point for deactivating the toxin. Here, we apply computational methods to uncover the allosteric mechanism of TcdB CPD and thereby advance the larger rational drug design project. Free energy calculations feature prominently in our toolkit. We use umbrella sampling to sample the large conformational transition of TcdB CPD and reproduce the allosteric effect on free energy surfaces. We also use free energy perturbation calculations to distinguish binding affinities among highly complex protonation states of the allosteric modulator phytate (IP6). Based on these and other computations, we construct a detailed allosteric mechanism of in the form of a switchable interaction network. This mechanism is thoroughly validated by both computations and experiments. We additionally contribute to the characterization of IP6 analogues with thiophosphate substitutions in the form of docking studies. Finally, we uncover the structural origins of the net loss of one proton on IP6–TcdB CPD complexation. Together, this computational treatment provides detailed structural and mechanistic insight on an important ligand-protein system.
