The symptoms of Clostridium difficile infections are caused by two exotoxins, 
TcdA and TcdB, which target host colonocytes by binding to unknown cell surface 
receptors, at least in part via their combined repetitive oligopeptide (CROP) 
domains. A combination of the anti-TcdA antibody actoxumab and the anti-TcdB 
antibody bezlotoxumab is currently under development for the prevention of 
recurrent C. difficile infections. We demonstrate here through various 
biophysical approaches that bezlotoxumab binds to specific regions within the 
N-terminal half of the TcdB CROP domain. Based on this information, we solved 
the x-ray structure of the N-terminal half of the TcdB CROP domain bound to Fab 
fragments of bezlotoxumab. The structure reveals that the TcdB CROP domain 
adopts a β-solenoid fold consisting of long and short repeats and that 
bezlotoxumab binds to two homologous sites within the CROP domain, partially 
occluding two of the four putative carbohydrate binding pockets located in TcdB. 
We also show that bezlotoxumab neutralizes TcdB by blocking binding of TcdB to 
mammalian cells. Overall, our data are consistent with a model wherein a single 
molecule of bezlotoxumab neutralizes TcdB by binding via its two Fab regions to 
two epitopes within the N-terminal half of the TcdB CROP domain, partially 
blocking the carbohydrate binding pockets of the toxin and preventing toxin 
binding to host cells.