INTERACTION OF CALCIUM WITH BORDETELLA-PERTUSSIS ADENYLATE-CYCLASE TOXIN
INTERACTION OF CALCIUM WITH BORDETELLA-PERTUSSIS ADENYLATE-CYCLASE TOXIN
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DOI：
10.1074/jbc.270.44.26370
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发表时间：
1995-11-03
影响因子：
4.8
通讯作者：
LADANT, D
中科院分区：
文献类型：
--
作者：
ROSE, T;SEBO, P;LADANT, D
腺苷酸本身的溶血活性与毒素的ATP循环催化活性无关，细胞毒性和溶血活性都是钙依赖性的。在这项工作中，我们分析了CyaA的钙相互作用特性。我们已经表明，CyaA暴露于氯化钙可以保留膜结合能力和溶血活性时，它进一步测定在过量的EGTA的存在下。对CyaA的钙含量的测定表明，在CyaA蛋白上仍有3-5个钙离子结合，表明存在高亲和力的钙结合位点，这些位点上的钙离子可能是毒素膜结合能力和溶血活性的必要条件。此外，CyaA具有大量（约45个）低亲和力（K-D = 0.5-0.8 mM）Ca 2+结合位点，其位于毒素的C末端，在氨基酸1007和1706之间。该区域主要由约45个GGXGXDXLX类型的重复序列组成（其中X代表任何氨基酸），其是RTX（重复：ToXin）细菌蛋白家族的特征。我们的数据表明，每一个可以结合一个钙离子，圆二色性光谱分析表明，钙结合到低亲和力网站诱导一个大的构象变化的CyaA，所揭示的α-螺旋结构的内容的重要增加。这种构象变化可能直接参与了CyaA催化结构域通过靶细胞质膜的Ca 2+依赖性转运。
The adenylate intrinsic hemolytic activity that is independent hom the ATP cycling catalytic activity of the toxin, Both the cytotoxic and hemolytic activities are calcium-dependent. In this work, we have analyzed the calcium interacting properties of CyaA. We have shown that CyaA exposed to CaCl2 could retain membrane binding capability and hemolytic 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 ions remained bound to the protein, suggesting the existence of Ca2+ binding sites of high affinity, finding of Ca2+ to these sites might be necessary for both the membrane binding capability and the hemolytic activity 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 located in the C terminus of the toxin, between amino acids 1007 and 1706. This region mainly consists of about 45 repeated sequences of the type GGXGXDXLX (where X represents any amino acid) that are characteristic of the RTX (Repeat: in ToXin) bacterial protein family. Our data suggest that each one can bind one calcium ion, Circular dichroism spectroscopy analysis showed that calcium binding to the low affinity sites induces a large conformational change of CyaA, as revealed by an important increase in the content of alpha-helical structures. This conformational change might be directly involved in the Ca2+-dependent translocation of the catalytic domain of CyaA through the plasma membrane of target cells.