Department of Chemistry, Wayne State University, Detroit, Michigan, United States of America.
PLoS One. 2012;7(7):e41518. doi: 10.1371/journal.pone.0041518. Epub 2012 Jul 23.
Clostridium difficile (C. difficile) is an opportunistic pathogen that can cause potentially lethal hospital-acquired infections. The cellular damage that it causes is the result of two large clostridial cytotoxins: TcdA and TcdB which act by glucosylating cytosolic G-proteins, mis-regulation of which induces apoptosis. TcdB is a large flexible protein that appears to undergo significant structural rearrangement upon accommodation of its substrates: UDP-glucose and a Rho-family GTPase. To characterize the conformational space of TcdB, we applied normal mode and hinge-region analysis, followed by long-timescale unbiased molecular dynamics. In order to examine the TcdB and RhoA interaction, macromolecular docking and simulation of the TcdB/RhoA complex was performed. Generalized Masked Delaunay analysis of the simulations determined the extent of significant motions. This combination of methods elucidated a wide range of motions within TcdB that are reiterated in both the low-cost normal mode analysis and the extensive MD simulation. Of particular interest are the coupled motions between a peripheral 4-helix bundle and a small loop in the active site that must rearrange to allow RhoA entry to the catalytic site. These extensive coupled motions are indicative of TcdB using a conformational capture mechanism for substrate accommodation.
艰难梭菌（C. difficile）是一种机会致病菌，可导致潜在致命的医院获得性感染。它引起的细胞损伤是由两种大的梭菌细胞毒素：TcdA 和 TcdB 引起的，这两种毒素通过糖基化细胞质 G 蛋白起作用，错误调节 G 蛋白会诱导细胞凋亡。TcdB 是一种大型灵活的蛋白质，在容纳其底物：UDP-葡萄糖和 Rho 家族 GTPase 时似乎会发生显著的结构重排。为了描述 TcdB 的构象空间，我们应用了正常模式和铰链区域分析，然后进行了长时间的无偏分子动力学模拟。为了研究 TcdB 和 RhoA 的相互作用，进行了大分子对接和 TcdB/RhoA 复合物的模拟。模拟的广义掩蔽 Delaunay 分析确定了显著运动的程度。这些方法的组合阐明了 TcdB 内广泛的运动，这些运动在低成本的正常模式分析和广泛的 MD 模拟中都得到了重复。特别值得注意的是，外周 4 螺旋束和活性部位小环之间的偶联运动，这种运动必须重新排列才能允许 RhoA 进入催化部位。这些广泛的偶联运动表明 TcdB 采用构象捕获机制来容纳底物。
