Structure of the cell-binding component of the <i>Clostridium difficile</i> binary toxin reveals a di-heptamer macromolecular assembly
Xingjian Xu, Raquel Godoy‐Ruiz, Kaylin A. Adipietro, Christopher Peralta, Danya Ben-Hail, Kristen M. Varney, Mary E. Cook, Braden M. Roth, Paul T. Wilder, Thomas E. Cleveland, Alexander Grishaev, Heather M. Neu, Sarah L. J. Michel, Wenbo Yu, Dorothy Beckett, Richard R. Rustandi, Catherine Lancaster, John W. Loughney, Adam Kristopeit, Sianny Christanti, Jessica W. Olson, Alexander D. MacKerell, Amédée des Georges, Edwin Pozharski, David J. Weber
Abstract
Targeting Clostridium difficile infection is challenging because treatment options are limited, and high recurrence rates are common. One reason for this is that hypervirulent C. difficile strains often have a binary toxin termed the C. difficile toxin, in addition to the enterotoxins TsdA and TsdB. The C. difficile 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 ( Sym CDTb; 3.14 Å) and an asymmetric form ( Asym CDTb; 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 Asym CDTb, a Ca 2+ 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 C. difficile .