Autocatalytic Processing of Clostridium difficile Toxin B BINDING OF INOSITOL HEXAKISPHOSPHATE

Clostridium difficile toxins A and B are major virulence factors responsible for induction of pseudomembranous colitis and antibiotic-associated diarrhea in men. The toxins possess a multidomain structure and only the N-terminal glucosyl-transferase domain, which inactivates Rho GTPases by glucosylation, is translocated into the cytosol of target cells. Processing of the toxin occurs by autocatalytic cleavage and is activated by inositol hexakisphosphate (InsP(6)). Here we studied the inherent protease activity in fragments of toxin B and determined the site of toxin B that interacts with InsP(6). We report that a fragment of toxin B, comprised of residues 1-955, is cleaved in the presence of InsP(6). In contrast, mutants of the catalytic triad of the putative cysteine protease domain did not cleave this fragment. [H-3] InsP(6) bound to holotoxin B and to the fragment 1-955, but not to a fragment comprising residues 900-2366 or the glucosyltransferase domain (residues 1-544). Binding to the putative cysteine protease domain (residues 544-955) was also observed. InsP(6)-binding was specific and saturable. Isothermal titration calorimetry revealed a K-d value of 2.4 mu M for binding of InsP(6) to toxin fragment 544-955 with a stoichiometry factor of 0.86. Lysine 600 of toxin B was identified as essential amino acid for InsP(6) binding and for InsP(6)-dependent activation of the protease activity.