Structures of a Conserved Type III Effector Domain

描述(由申请人提供)：许多动植物病原菌，包括NIAID优先病原菌名单上的至少七种，通过III型分泌(TTS)系统将毒力蛋白(效应物)直接输送到宿主细胞。在那里，它们干扰细胞信号来操纵细胞，以利于细菌。这项建议调查了特定的效应蛋白如何被TTS设备识别以进行随后的分泌和转位的结构决定因素。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非保守区，以确保有效的易位。有趣的是，在鼠伤寒沙门氏菌中，一组9个效应器具有同源的氨基末端结构域(约145个残基，称为‘WEKIF’结构域)，但在其他地方是无关的。这些结构域是移位所必需的，也是足够的；没有发现分泌伴侣，也可能不需要它们。该结构域的保守性表明，它还可能将效应器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：涉及这个领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。这里提出了对单个WEKIF结构域的比较结构研究。拟议研究的目标1将改进SspH1效应器WEKIF结构域的现有晶体，并开始晶体结构测定。目的2建议表达和纯化另外四个WEKIF结构域，SIRP、SifA、SSEL和SseJ，并对结晶条件进行筛选。根据一个相关但积极的结果，Aim 3将筛选全长SspH1，其中包含富含亮氨酸的重复效应结构域，以寻找晶体。

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.