INTERACTION OF CALCIUM WITH BORDETELLA-PERTUSSIS ADENYLATE-CYCLASE TOXIN

The adenylate intrinsic hemolytic activity that is independent hom the ATP cycling catalytic activity of the toxin, Both the cytotoxic and h emolytic activities are calcium-dependent. In this work, we have analy zed the calcium interacting properties of CyaA. We have shown that Cya A exposed to CaCl2 could retain membrane binding capability and hemoly tic activity when it was further assayed in the presence of an excess of EGTA. Determination of the calcium content of CyaA exposed first to calcium and subsequently to EGTA indicated that some (3-5) calcium io ns remained bound to the protein, suggesting the existence of Ca2+ bin ding sites of high affinity, finding of Ca2+ to these sites might be n ecessary for both the membrane binding capability and the hemolytic ac tivity of the toxin. In addition, CyaA possesses a large number (about 45) of low affinity (K-D = 0.5-0.8 mM) Ca2+ binding sites that are lo cated in the C terminus of the toxin, between amino acids 1007 and 170 6. This region mainly consists of about 45 repeated sequences of the t ype GGXGXDXLX (where X represents any amino acid) that are characteris tic of the RTX (Repeat: in ToXin) bacterial protein family. Our data s uggest that each one can bind one calcium ion, Circular dichroism spec troscopy analysis showed that calcium binding to the low affinity site s induces a large conformational change of CyaA, as revealed by an imp ortant increase in the content of alpha-helical structures. This confo rmational change might be directly involved in the Ca2+-dependent tran slocation of the catalytic domain of CyaA through the plasma membrane of target cells.