Institut Pasteur, Unité de Biochimie des Interactions Macromoléculaires, CNRS URA 2185, Département de Biologie Structurale et Chimie, 25-28 rue du Dr. Roux, 75724 Paris Cedex 15, France.
Biochemistry. 2010 Jan 19;49(2):318-28. doi: 10.1021/bi9016389.
Bordetella pertussis, the causative agent of whooping cough, secretes among various toxins an adenylate cyclase (CyaA) that displays a unique mechanism of cell invasion, which involves a direct translocation of its N-terminal catalytic domain (AC, 400 residues) across the plasma membrane of the eukaryotic targeted cells. Once into the cytosol, AC is activated by endogenous calmodulin and produces toxic amounts of cAMP. The structure of AC in complex with the C-terminal part of calmodulin has recently been determined. However, as the structure of the catalytic domain in the absence of calmodulin is still lacking, the molecular basis of AC activation by calmodulin remains largely unknown. To characterize this activation mechanism, we investigated here the biophysical properties of the isolated catalytic domain in solution with or without calmodulin. We found that calmodulin triggered only minor modifications of the protein secondary and tertiary structure but had a pronounced effect on the hydrodynamic properties of AC. Indeed, while the isolated catalytic domain was spherical and hydrated, it underwent a significant elongation as well as compaction and dehydration upon calmodulin interaction. On the basis of these data, we propose a model for the structural transition between the calmodulin-free and calmodulin-bound AC.
百日咳博德特氏菌是百日咳的病原体，它分泌多种毒素，其中包括一种腺苷酸环化酶（CyaA），该酶具有独特的细胞入侵机制，涉及其 N 端催化结构域（AC，400 个残基）直接穿过真核靶细胞的质膜的易位。一旦进入细胞质，AC 就被内源性钙调蛋白激活，并产生大量有毒的 cAMP。最近已经确定了 AC 与钙调蛋白 C 端部分复合物的结构。然而，由于缺乏钙调蛋白的催化结构域的结构仍然缺失，因此钙调蛋白激活 AC 的分子基础在很大程度上仍然未知。为了表征这种激活机制，我们在此研究了有无钙调蛋白存在时溶液中分离的催化结构域的生物物理特性。我们发现，钙调蛋白仅触发了蛋白质二级和三级结构的微小修饰，但对 AC 的水动力性质有显著影响。事实上，虽然分离的催化结构域是球形且水合的，但在与钙调蛋白相互作用时，它会发生显著的伸长、紧凑和去水合。基于这些数据，我们提出了钙调蛋白游离和结合的 AC 之间结构转变的模型。
