Structures of a Conserved Type III Effector Domain

描述（由申请人提供）：许多动植物致病菌，包括NIAID优先病原体清单上的至少7种，通过III型分泌（TTS）系统将毒力蛋白（效应器）直接传递到宿主细胞中。在那里，它们破坏细胞信号，为细菌的利益操纵细胞。本研究探讨了TTS装置如何识别特异性效应蛋白以进行随后的分泌和易位的结构决定因素。鼠伤寒沙门氏菌是一种B类病原体，可引起人和牲畜严重腹泻。对于一些效应器，一个小的伴侣蛋白结合到效应器氨基末端附近的一个非保守区域，以确保有效的易位。有趣的是，在鼠伤寒沙门氏菌中，一组9个效应物具有同源的氨基末端结构域（约145个残基，称为“WEKIF”结构域），但在其他地方不相关。这些结构域是易位所必需和充分的；没有发现分泌伴侣，可能也不需要。该结构域的保守性表明，它也可能将效应物定位到宿主细胞中的特定区室或蛋白质上。该研究的长期目标是确定蛋白质的结构基础：涉及该结构域的蛋白质相互作用。这是发现抗菌药物开发潜在靶点的第一步。本文建议对各个WEKIF域进行比较结构研究。拟研究的目的1将改进SspH1效应体WEKIF结构域的现有晶体，并开始晶体学结构测定。目的2提出表达和纯化其他四个WEKIF结构域SIrP、SifA、Ssel和SseJ，并筛选结晶条件。在一个相关但阳性的结果之后，Aim 3将筛选包含富含亮氨酸的重复效应域的全长SspH1晶体。

DESCRIPTION (provided by applicant): Many animal and plant pathogenic bacteria, including at least seven on the NIAID priority pathogen list, deliver virulence proteins (effectors) directly into host cells through type III secretion (TTS) systems. There they disrupt cell signaling to manipulate the cell for the bacteria's advantage. This proposal investigates the structural determinants of how specific effector proteins are recognized by the TTS apparatus for subsequent secretion and translocation. Salmonella typhimurium is a class B pathogen that causes severe diarrhea in people and livestock. For some effectors, a small chaperone protein binds to a non-conserved region near the amino-terminus of the effector to ensure efficient translocation. Interestingly, in S. typhimurium, a set of nine effectors have homologous amino-terminal domains (about 145 residues, termed 'WEKIF' domains) but are unrelated elsewhere. These domains are required and sufficient for translocation; no secretion chaperones have been identified and they may not be required. The conserved nature of the domain suggests that it also may localize the effector to specific compartments or proteins in the host cell. The long-term goal of the research is to define the structural basis for the protein:protein interactions involving this domain. This is the first step for discovering potential targets for antimicrobial development. Comparative structural studies of the individual WEKIF domains are proposed here. Aim 1 of the proposed research will improve existing crystals of the WEKIF domain of the SspH1 effector, and begin crystallographic structure determination. Aim 2 proposes to express and purify four other WEKIF domains, SIrP, SifA, Ssel, and SseJ, and screen for crystallization conditions. Following up on a related, but positive result, Aim 3 will screen the full-length SspH1, containing the leucine-rich repeat effector domains, for crystals.