Structures of a Conserved Type III Effector Domain

描述（由申请人提供）：许多动物和植物病原菌，包括NIAID优先病原体列表中的至少7种，通过III型分泌（TTS）系统将毒力蛋白（效应子）直接递送到宿主细胞中。 在那里，它们破坏细胞信号，操纵细胞为细菌的优势。 这个建议调查的结构决定因素如何特定的效应蛋白被认可的TTS装置随后的分泌和易位。 鼠伤寒沙门氏菌是一种B类病原体，可导致人和牲畜严重腹泻。 对于一些效应物，小分子伴侣蛋白结合到效应物的氨基末端附近的非保守区域以确保有效的易位。 有趣的是，在S.在鼠伤寒沙门氏菌中，一组9个效应子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的，并且足以易位;尚未鉴定出分泌分子伴侣，并且它们可能不是必需的。 该结构域的保守性质表明它也可以将效应子定位于宿主细胞中的特定隔室或蛋白质。 这项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。 这是发现抗菌剂开发潜在靶点的第一步。 在这里提出的个别WEKIF域的比较结构研究。 所提出的研究的目的1将改进SspH 1效应器的WEKIF结构域的现有晶体，并且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。 在一个相关的，但积极的结果，目标3将筛选全长SspH 1，含有富含亮氨酸的重复效应域，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.