Structure of the cell-binding component of the Clostridium difficile binary toxin reveals a novel macromolecular assembly

Therapeutic approaches for targeting Clostridium difficile infection (CDI) are challenging because treatment options are limited and there are unacceptably high recurrence rates, particularly with hypervirulent strains (i.e. NAP1/P1/027). One reason for this problem is that increasing occurrences of hypervirulent CDI has a binary toxin termed the C. difficile toxin (CDT), in addition to the enterotoxins TsdA and TsdB. The CDT binary toxin has an enzymatic component, termed CDTa, and a pore-forming or delivery subunit termed CDTb. We characterized CDTb at the molecular level using a combination of single particle cryo-EM, X-ray crystallography, NMR, and other biophysical methods. These studies uncovered that two novel di-heptamer structures occur for CDTb in the absence of CDTa. This includes a symmetric form (SymCDTb), solved here at 3.14 A, and an asymmetric form (AsymCDTb), solved at 2.84 A, respectively, via single particle cryoEM. Unlike any other binary toxin observed to date, the two heptamer units of SymCDTb and AsymCDTb are assembled via a unique sequence at the C-terminus of CDTb, termed the receptor binding domain 2 (RBD2). The roles played by two receptor-binding domains of CDTb were of specific interest since one (RBD1) lacked sequence homology to any other known toxin and the other (RBD2) was at the C-terminus of CDTb and is absent in other well studied heptameric toxins (i.e. anthrax). Importantly, a novel Ca2+ binding site was discovered in RBD1 that is important for its stability, and RBD2 was found to be critical for establishing the two novel di-heptamer macromolecular assemblies observed for CDTb, which is necessary for host cell toxicity. Together, these and other regions of CDTb can be considered in future structure-based drug-discovery strategies for targeting CDT in the most severe strains of CDI.