Crystallographical and molecular biological analysis of ia component of C. perfringens iota-toxin

产气荚膜梭菌adp -核苷化非肌（β/γ）肌动蛋白和骨骼肌α-肌动蛋白。在这里，我们在1.8Å上展示了Ia与NADH配合物的晶体结构。研究发现Ia可分为两个结构域，称为n结构域和c结构域，c结构域具有结合NADH的空腔。基于该结构的定点诱变表明，Ia的催化机制是通过Snl型反应进行的：Arg295和Arg352的正电荷原子分别与NAD^+的α-和β-磷酸基团电相互作用，Glu380固定了核糖位置，Phe349的苯环限制了烟酰胺环的旋转，烟酰胺羧胺与Arg296的主链酰胺和羰基形成氢键。NAD^+与Ia的特异性结合诱导了NAD^+的构象变化，导致配合物中NAD的环状构象。α-磷酸基团可能是snl型反应中最重要的裂解基团，它与烟酰胺的氨基（距离2.92Å）形成氢键，然后将电子从氨基上抽离。因此氢键的形成诱导了n -糖苷键的自发断裂。adp -核糖基部分的氧碳离子应该通过与带负电荷的Glu378羧酸基的螯合来稳定。在Ia-actin复合物形成时，肌动蛋白的Arg177位于氧碳阳离子附近。然后adp核糖组转移到肌动蛋白的Arg177上。

The iotaa component (ia) of Clostridium perfringens ADP-ribosylates nonmuscld (β/γ actin and skeletal muscle α-actin. Here, we showed crystal structures of Ia complex with NADH at 1.8Å. It was found that Ia can be divided into two domains, termed the N-domain and the C-domain that has a cavity which binds NADH. A site-directed mutagenesis based on the structure suggested that the catalytic mechanism of Ia proceeds via Snl type reaction as follows ; the positively charged atoms from Arg295 and Arg352 electrically interact with α- and β-phosphate groups of NAD^+, respectively, Glu380 fixes the ribose position, the benzene ring of Phe349 restricts the nicotinamide ring rotation and the nicotinamide carboxyamide forms hydrogen bonds with the main chain amide and carbonyl of Arg296.The specific binding of NAD^+ to Ia induces a conformational change in NAD^+ that results in a ring-like conformation of NAD in the complex. The α-phosphate group could be most important for cleavage via Snl-type reaction, by making a hydrogen bond with the amino group of nicotinamide (distance of 2.92Å) and then withdrawing the electron from the amino group. Thus the hydrogen bond formation induces the spontaneous cleavage of N-glycoside bond. The resulting oxocarbonium cation of the ADP-ribosyl moiety should be stabilized by chelation with the negatively charged carboxylate group of Glu378.The Arg177 of actin is located near the oxocarbonium cation upon Ia-actin complex formation. Then the ADP-ribose group is transferred to the Arg177 of actin.