Secretin Architecture

 描述（由申请人提供）：促胰液素是一种同源多聚体外膜通道，是IV型菌毛（T4P）、2型分泌和3型分泌系统唯一共有的蛋白质。因此，分泌素在毒力因子中享有独特的突出地位。虽然几个周质分泌素域的结构已被解决的X射线晶体学，和分泌素的整体形状已被描述的电子显微镜，足够的细节，以了解机制，使分泌素相互作用的其他组成部分，其各自的机器，并打开选择性地允许通过其基板尚未实现。在这个提议中，我们描述了我们将如何推进我们对胰泌素功能的理解。使用各种技术，包括半胱氨酸诱变和Förster共振能量转移，我们描述了一个拓扑结构模型BfpB，分泌素从T4P的肠致病性大肠杆菌。这项建议的第一个目的是 在此基础上，通过使用体内二硫键交联来捕获分泌素中的菌毛蛋白，从而绘制分泌素通道的壁。低温电子显微镜的最新进展允许在接近原子范围的分辨率的大型复合物的结构分析。在目标二中，我们将利用这些进展，以前所未有的细节来可视化与之相关的胰泌素和蛋白质。本提案中描述的实验将提供关键的结构信息，这对于我们完整理解T4P生物发生的长期目标至关重要。此外，这些信息也将对分泌系统产生影响，并可能最终对新疗法产生实际影响。

 DESCRIPTION (provided by applicant): The secretin, a homomultimeric outer membrane channel, is the only protein common to type IV pilus (T4P), type 2 secretion and type 3 secretion systems. Therefore, secretins enjoy a unique position of prominence among virulence factors. Although the structures of several periplasmic secretin domains have been solved by X-ray crystallography, and the overall shape of secretins has been described by electron microscopy, sufficient detail to understand the mechanism that allows secretins to interact with other components of their respective machines and to open selectively to allow passage of their substrates has not be achieved. In this proposal we describe how we will advance our understanding of secretin function. Using a variety of techniques, including cysteine mutagenesis and Förster resonance energy transfer, we described a topology model for BfpB, the secretin from the T4P of enteropathogenic Escherichia coli. The first aim of this proposal will build on that knowledge by using in vivo disulfide cross-linking to trap the pilin in the secretin and thereby map the walls of the secretin channel. Recent advances in cryo-electron microscopy have permitted the structural analysis of large complexes at resolutions approaching the atomic range. In aim two we will capitalize on these advances to visualize the secretin and proteins with which it associates in unprecedented detail. The experiments described in this proposal will provide critical structural information that is essential for our lng-term goal of a complete understanding of T4P biogenesis. Furthermore, this information will also have implications for secretion systems and may ultimately have practical implications for new therapeutics.