Clostridium difficile is an opportunistic pathogen that establishes in the colon when the gut microbiota is disrupted by antibiotics or disease. C. difficile infection (CDI) is largely caused by two virulence factors TcdA and TcdB. Here, we report a 3.87 Å resolution crystal structure of TcdB holotoxin that captures a unique conformation of TcdB at endosomal pH. Complementary biophysical studies suggest that the CROPs domain of TcdB is dynamic and can sample open and closed conformations that may facilitate modulation of TcdB activity in response to environmental and cellular cues during intoxication. Furthermore, we report three crystal structures of TcdB–antibody complexes that reveal how antibodies could specifically inhibit the activities of individual TcdB domains. Our studies provide novel insights into the structure and function of TcdB holotoxin and identify intrinsic vulnerabilities that could be exploited to develop new therapeutics and vaccines for the treatment of CDI.

Botulinum neurotoxin E (BoNT/E) is one of the major causes of human botulism and paradoxically also a promising therapeutic agent. Here we determined the co-crystal structures of the receptor-binding domain of BoNT/E (HCE) in complex with its neuronal receptor synaptic vesicle glycoprotein 2A (SV2A) and a nanobody that serves as a ganglioside surrogate. These structures reveal that the protein-protein interactions between HCE and SV2 provide the crucial location and specificity information for HCE to recognize SV2A and SV2B, but not the closely related SV2C. At the same time, HCE exploits a separated sialic acid-binding pocket to mediate recognition of an N-glycan of SV2. Structure-based mutagenesis and functional studies demonstrate that both the protein-protein and protein-glycan associations are essential for SV2A-mediated cell entry of BoNT/E and for its potent neurotoxicity. Our studies establish the structural basis to understand the receptor-specificity of BoNT/E and to engineer BoNT/E variants for new clinical applications.