Robien Mark A, Krumm Brian E, Sandkvist Maria, Hol Wim G J
Department of Biochemistry, Biomolecular Structure Center, University of Washington, P.O. Box 357742, Seattle, WA 98195, USA.
J Mol Biol. 2003 Oct 24;333(3):657-74. doi: 10.1016/j.jmb.2003.07.015.
Type II secretion systems consist of an assembly of 12-15 Gsp proteins responsible for transporting a variety of virulence factors across the outer membrane in several pathogenic bacteria. In Vibrio cholerae, the major virulence factor cholera toxin is secreted by the Eps Type II secretion apparatus consisting of 14 Eps proteins. One of these, EpsE, is a cytoplasmic putative NTPase essential for the functioning of the Eps system and member of the GspE subfamily of Type II secretion ATPases. The crystal structure of a truncated form of EpsE in nucleotide-liganded and unliganded state has been determined, and reveals a two-domain architecture with the four characteristic sequence "boxes" of the GspE subfamily clustering around the nucleotide-binding site of the C-domain. This domain contains two C-terminal subdomains not reported before in this superfamily of NTPases. One of these subdomains contains a four-cysteine motif that appears to be involved in metal binding as revealed by anomalous difference density. The EpsE subunits form a right-handed helical arrangement in the crystal with extensive and conserved contacts between the C and N domains of neighboring subunits. Combining the most conserved interface with the quaternary structure of the C domain in a distant homolog, a hexameric model for EpsE is proposed which may reflect the assembly of this critical protein in the Type II secretion system. The nucleotide ligand contacts both domains in this model. The N2-domain-containing surface of the hexamer appears to be highly conserved in the GspE family and most likely faces the inner membrane interacting with other members of the Eps system.
II型分泌系统由12 - 15种Gsp蛋白组成，负责在几种致病细菌中跨外膜转运多种毒力因子。在霍乱弧菌中，主要毒力因子霍乱毒素由由14种Eps蛋白组成的Eps II型分泌装置分泌。其中之一EpsE是一种胞质推定NTPase，对Eps系统的功能至关重要，是II型分泌ATPases的GspE亚家族成员。已确定了核苷酸结合和未结合状态下截短形式的EpsE的晶体结构，揭示了一种双结构域结构，GspE亚家族的四个特征序列“框”聚集在C结构域的核苷酸结合位点周围。该结构域包含两个此前在这个NTPases超家族中未报道过的C末端亚结构域。其中一个亚结构域包含一个四半胱氨酸基序，异常差分密度显示该基序似乎参与金属结合。EpsE亚基在晶体中形成右手螺旋排列，相邻亚基的C结构域和N结构域之间有广泛且保守的接触。结合最保守的界面与远亲同源物中C结构域的四级结构，提出了EpsE的六聚体模型，该模型可能反映了这种关键蛋白在II型分泌系统中的组装。在该模型中，核苷酸配体与两个结构域都有接触。六聚体含N2结构域的表面在GspE家族中似乎高度保守，很可能面向内膜与Eps系统的其他成员相互作用。
