Abendroth Jan, Bagdasarian Michael, Sandkvist Maria, Hol Wim G J
Department of Biochemistry, Biomolecular Structure Center, School of Medicine, University of Washington, P.O. Box 357742, Seattle, WA 98195-7242, USA.
J Mol Biol. 2004 Nov 26;344(3):619-33. doi: 10.1016/j.jmb.2004.09.062.
The type II secretion system (T2SS) is used by several Gram-negative bacteria for the secretion of hydrolytic enzymes and virulence factors across the outer membrane. In these secretion systems, a complex of 12-15 so-called "Gsp proteins" spans from a regulatory ATPase in the cytoplasm, via several signal or energy transducing proteins in the inner membrane and the pseudopilins in the periplasm, to the actual pore in the outer membrane. The human pathogen Vibrio cholerae employs such an assembly, called the Eps system, for the export of its major virulence factor, cholera toxin, from its periplasm into the lumen of the gastro-intestinal tract of the host. Here, we report the atomic structure of the major cytoplasmic domain of the inner membrane-spanning EpsL protein from V. cholerae. EpsL is the binding partner of the regulatory ATPase EpsE as well as of EpsM and pseudopilins, and is therefore a critical link between the cytoplasmic and the periplasmic part of the Eps-system. The 2.7A resolution structure was determined by a combination of Se-Met multiple anomalous dispersion (MAD) and multiple isomorphous replacement with anomalous scattering (MIRAS) phasing methods. The 28kDa cytoplasmic domain of EpsL (cyto-EpsL) consists of three beta-sheet-rich domains. With domains I and III similar to the RNaseH-fold, cyto-EpsL unexpectedly shows structural homology with the superfamily of actin-like ATPases. cyto-EpsL, however, is an unusual member of this superfamily as it misses the canonical actin domains 1B and 2B, which are common yet variable in this superfamily. Moreover, cyto-EpsL has an additional domain II, which has the topology of an SHS2-fold module. Within the superfamily this fold module has been observed only for domain 1C of the cell division protein FtsA, in which it mediates protein-protein interactions. This domain II displays great flexibility and contributes to a pronounced negatively charged canyon on the surface of cyto-EpsL. Functional data as well as structural homology and sequence conservation suggest that domain II interacts with EpsE, the major cytoplasmic binding partner of EpsL.
II型分泌系统（T2SS）被多种革兰氏阴性菌用于将水解酶和毒力因子分泌到外膜之外。在这些分泌系统中，一个由12 - 15个所谓的“Gsp蛋白”组成的复合体从细胞质中的调节性ATP酶出发，经过内膜中的几个信号或能量转导蛋白以及周质中的假菌毛蛋白，一直延伸到外膜中的实际孔道。人类病原体霍乱弧菌利用这样一种被称为Eps系统的组装结构，将其主要毒力因子霍乱毒素从周质输出到宿主胃肠道腔中。在此，我们报道了霍乱弧菌跨内膜EpsL蛋白主要细胞质结构域的原子结构。EpsL是调节性ATP酶EpsE以及EpsM和假菌毛蛋白的结合伴侣，因此是Eps系统细胞质部分和周质部分之间的关键连接。通过硒代甲硫氨酸多波长反常散射（MAD）和带有反常散射的多同晶置换（MIRAS）相位法相结合，确定了分辨率为2.7埃的结构。EpsL的28kDa细胞质结构域（cyto - EpsL）由三个富含β - 折叠的结构域组成。由于结构域I和III与核糖核酸酶H折叠相似，cyto - EpsL意外地与肌动蛋白样ATP酶超家族显示出结构同源性。然而，cyto - EpsL是这个超家族中的一个不寻常成员，因为它缺少该超家族中常见但可变的典型肌动蛋白结构域1B和2B。此外，cyto - EpsL还有一个额外的结构域II，其拓扑结构为SHS2 - 折叠模块。在这个超家族中，这种折叠模块仅在细胞分裂蛋白FtsA的结构域1C中被观察到，在那里它介导蛋白质 - 蛋白质相互作用。这个结构域II表现出极大的灵活性，并在cyto - EpsL表面形成了一个明显带负电荷的峡谷。功能数据以及结构同源性和序列保守性表明，结构域II与EpsL的主要细胞质结合伴侣EpsE相互作用。
