Secretin Architecture

 描述(申请人提供)：分泌素是一种同源多聚体外膜通道，是IV型菌毛(T4P)、2型分泌系统和3型分泌系统共有的唯一蛋白质。因此，分泌素在毒力因子中具有独特的突出地位。虽然X-射线结晶学已经解决了几个周质分泌素结构域的结构，电子显微镜已经描述了分泌素的整体形状，但还没有足够的细节来理解分泌素与各自机器的其他组件相互作用的机制，以及选择性地打开分泌素以允许底物通过的机制。在这项提议中，我们描述了我们将如何推进对分泌素功能的理解。利用半胱氨酸诱变和Förster共振能量转移等多种技术，我们描述了肠源性大肠杆菌T4P的分泌素BfpB的拓扑模型。这项提议的第一个目标是 在此基础上，通过在体内使用二硫化物交联物来捕获分泌素中的菌毛素，从而绘制出分泌素通道的壁图。低温电子显微镜的最新进展使得能够以接近原子范围的分辨率对大型络合物进行结构分析。在第二个目标中，我们将利用这些进展，以前所未有的细节可视化与之相关的分泌素和蛋白质。本提案中描述的实验将提供关键的结构信息，这些信息对于我们全面了解T4P生物发生的液化天然气长期目标至关重要。此外，这些信息还将对分泌系统产生影响，并最终可能对新的治疗方法产生实际影响。

 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.