Korotkov Konstantin V, Krumm Brian, Bagdasarian Michael, Hol Wim G J
Department of Biochemistry, Biomolecular Structure Center, University of Washington, Box 357742, Seattle, WA 98195, USA.
J Mol Biol. 2006 Oct 20;363(2):311-21. doi: 10.1016/j.jmb.2006.08.037. Epub 2006 Aug 18.
The type 2 secretion system (T2SS) occurring in Gram-negative bacteria is composed of 12-15 different proteins which form large assemblies spanning two membranes and secreting several virulence factors in folded state across the outer membrane. The T2SS component EpsC of Vibrio cholerae plays an important role in this machinery. While anchored in the inner membrane, by far the largest part of EpsC is periplasmic, containing a so-called homology region (HR) domain and a PDZ domain. Here we report studies on the structure and function of both periplasmic domains of EpsC. The crystal structures of two variants of the PDZ domain of EpsC from V. cholerae were determined at better than 2 A resolution. Compared to the short variant, the longer variant contains an additional N-terminal helix, and reveals a significant difference in the position of helix alphaB with respect to the beta-sheet. Both our structures show that the PDZ domain of EpsC adopts a more open form than in previously reported structures of other PDZ domains. Most interestingly, in the crystals of the short EpsC-PDZ domain the peptide binding groove interacts with an alpha-helix from a neighboring subunit burying approximately 921 A2 solvent accessible surface. This makes it possible that the PDZ domain of this bacterial protein binds proteins in a manner which is altogether different from that seen in any other PDZ domain so far. We also determined that the HR domain of EpsC is primarily responsible for the interaction with the secretin EpsD, while the PDZ is not, or much less, so. This new finding, together with studies of others, leads to the suggestion that the PDZ domain of EpsC may interact with exoproteins to be secreted while the HR domain plays a key role in linking the inner-membrane sub-complex of the T2SS in V. cholerae to the outer membrane secretin.
革兰氏阴性菌中的2型分泌系统（T2SS）由12 - 15种不同蛋白质组成，这些蛋白质形成跨越两个膜的大型组装体，并以折叠状态将多种毒力因子分泌穿过外膜。霍乱弧菌的T2SS组分EpsC在这一机制中发挥重要作用。EpsC大部分锚定在内膜中，但其绝大部分位于周质，包含一个所谓的同源区域（HR）结构域和一个PDZ结构域。在此，我们报告了对EpsC两个周质结构域的结构和功能的研究。霍乱弧菌EpsC的PDZ结构域的两个变体的晶体结构在优于2 Å的分辨率下得以确定。与短变体相比，长变体包含一个额外的N端螺旋，并且αB螺旋相对于β折叠的位置存在显著差异。我们的两个结构均表明，EpsC的PDZ结构域比先前报道的其他PDZ结构域采用更开放的形式。最有趣的是，在短EpsC - PDZ结构域的晶体中，肽结合槽与来自相邻亚基的α螺旋相互作用，掩埋了约921 Ų的溶剂可及表面。这使得这种细菌蛋白的PDZ结构域可能以与迄今所见的任何其他PDZ结构域完全不同的方式结合蛋白质。我们还确定，EpsC的HR结构域主要负责与分泌素EpsD相互作用，而PDZ结构域则不然，或者作用小得多。这一新发现与其他研究结果共同表明，EpsC的PDZ结构域可能与待分泌的外蛋白相互作用，而HR结构域在将霍乱弧菌T2SS的内膜亚复合体与外膜分泌素连接起来方面发挥关键作用。
