Protective antigen (PA), the receptor-binding moiety of anthrax toxin, contains two calcium atoms buried within domain 1' (amino acid residues 168-258). We showed that these ions are stably bound and exchange with free Ca-45(2+) only slowly (t(1/2) similar to 4.0 h). Dissociation is the rate-limiting step. PA(63), the heptameric prepore form of PA. showed a slightly higher exchange rate than the monomeric intact protein. Exchange by this form was retarded by binding of the enzymatic moieties of the toxin, but was unaffected by reducing the pH to 5.0, a condition known to trigger conversion of the prepore to the pore form. These results are consistent with the hypothesis that bound Ca2+ within PA plays primarily a structural role, maintaining domain P in a conformation that allows PA(63) to oligomerize and bind the enzymatic moieties of the toxin. (C) 2002 Elsevier Science (USA). All rights reserved.