Structure of the cell-binding component of the <i>Clostridium difficile</i> binary toxin reveals a di-heptamer macromolecular assembly

Abstract

Targeting <i>Clostridium difficile</i> infection is challenging because treatment options are limited, and high recurrence rates are common. One reason for this is that hypervirulent <i>C. difficile</i> strains often have a binary toxin termed the <i>C. difficile</i> toxin, in addition to the enterotoxins TsdA and TsdB. The <i>C. difficile</i> toxin has an enzymatic component, termed CDTa, and a pore-forming or delivery subunit termed CDTb. CDTb was characterized here using a combination of single-particle cryoelectron microscopy, X-ray crystallography, NMR, and other biophysical methods. In the absence of CDTa, 2 di-heptamer structures for activated CDTb (1.0 MDa) were solved at atomic resolution, including a symmetric (^SymCDTb; 3.14 Å) and an asymmetric form (^AsymCDTb; 2.84 Å). Roles played by 2 receptor-binding domains of activated CDTb were of particular interest since the receptor-binding domain 1 lacks sequence homology to any other known toxin, and the receptor-binding domain 2 is completely absent in other well-studied heptameric toxins (i.e., anthrax). For ^AsymCDTb, a Ca²⁺ binding site was discovered in the first receptor-binding domain that is important for its stability, and the second receptor-binding domain was found to be critical for host cell toxicity and the di-heptamer fold for both forms of activated CDTb. Together, these studies represent a starting point for developing structure-based drug-design strategies to target the most severe strains of <i>C. difficile</i>.