Kawahara Kazuki, Yonogi Shinya, Munetomo Ryota, Oki Hiroya, Yoshida Takuya, Kumeda Yuko, Matsuda Shigeaki, Kodama Toshio, Ohkubo Tadayasu, Iida Tetsuya, Nakamura Shota
Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka, Japan.
Osaka Prefectural Institute of Public Health, Osaka, Osaka, Japan; Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.
Biochem Biophys Res Commun. 2016 Nov 11;480(2):261-267. doi: 10.1016/j.bbrc.2016.10.042. Epub 2016 Oct 15.
Binary enterotoxin of Clostridium perfringens (BEC), consisting of the components BECa and BECb, was recently identified as a novel enterotoxin produced by C. perfringens that causes acute gastroenteritis in humans. Although the detailed mechanism of cell intoxication by BEC remains to be defined, BECa shows both NAD-glycohydrolase and actin ADP-ribosyltransferase activities in the presence of NAD. In this study, we determined the first crystal structure of BECa in its apo-state and in complex with NADH. The structure of BECa shows striking resemblance with other binary actin ADP-ribosylating toxins (ADPRTs), especially in terms of its overall protein fold and mechanisms of substrate recognition. We present a detailed picture of interactions between BECa and NADH, including bound water molecules located near the C1'-N glycosidic bond of NADH and the catalytically important ADP-ribosylating turn-turn (ARTT) loop. We observed that the conformational rearrangement of the ARTT loop, possibly triggered by a conformational change involving a conserved tyrosine residue coupled with substrate binding, plays a crucial role in catalysis by properly positioning a catalytic glutamate residue in the E-X-E motif of the ARTT loop in contact with the nucleophile. Our results for BECa provide insight into the common catalytic mechanism of the family of binary actin ADPRTs.
产气荚膜梭菌的二元肠毒素（BEC）由BECa和BECb组成，最近被鉴定为产气荚膜梭菌产生的一种新型肠毒素，可导致人类急性肠胃炎。尽管BEC导致细胞中毒的详细机制尚待确定，但在烟酰胺腺嘌呤二核苷酸（NAD）存在的情况下，BECa表现出烟酰胺腺嘌呤二核苷酸糖水解酶和肌动蛋白ADP核糖基转移酶活性。在本研究中，我们确定了BECa处于脱辅基状态以及与还原型辅酶I（NADH）结合时的首个晶体结构。BECa的结构与其他二元肌动蛋白ADP核糖基化毒素（ADPRT）具有显著相似性，尤其是在其整体蛋白质折叠和底物识别机制方面。我们展示了BECa与NADH之间相互作用的详细情况，包括位于NADH的C1'-N糖苷键附近的结合水分子以及具有催化重要性的ADP核糖基化转折环（ARTT环）。我们观察到，ARTT环的构象重排可能由涉及一个保守酪氨酸残基的构象变化与底物结合共同触发，通过将ARTT环E-X-E模体中的催化谷氨酸残基正确定位以接触亲核试剂，在催化过程中发挥关键作用。我们对BECa的研究结果为二元肌动蛋白ADPRT家族的共同催化机制提供了见解。
