T. Rose et al., INTERACTION OF CALCIUM WITH BORDETELLA-PERTUSSIS ADENYLATE-CYCLASE TOXIN, The Journal of biological chemistry, 270(44), 1995, pp. 26370-26376
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.