Structure of ToxR/S and TcpP/H: Virulence Regulators

ToxR/S 和 TcpP/H 的结构：毒力调节剂

• 批准号: 6708909
• 负责人: Victor J. DiRita
• 金额: $ 7.65万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6708909
• 关键词: DNA binding protein; Vibrio cholerae; bacterial proteins; binding sites; cholera toxin; crystallization; gene expression; genetic transcription; membrane proteins; method development; pilus; protein binding; protein protein interaction; protein purification; protein structure function; virulence

DESCRIPTION (provided by applicant): This proposal seeks to develop methods for overexpressing, purifying and crystallizing membrane-localized transcription control proteins from Vibrio cholerae, the agent of human cholera disease. ToxR and TcpP are bi-topic membrane proteins with cytoplasmic DNA binding/activation domains. They control expression of important virulence factors by virtue of their ability to activate expression of toxT, the activator of genes encoding cholera toxin and toxin-coregulated pilus. The DNA binding/activation domains of ToxR and TcpP are homologous to the winged Helix-Turn-Helix (w-HTH) family of proteins. The structures of other w-HTH domains has been solved, but the unusual membrane topology of ToxR and TcpP, and significant preliminary data aimed at determining how they recognize DNA and activate toxT transcription, compel an interest in solving their crystal structures when bound to operator DNA. Upon completion, this study will enable us to discriminate between distinct hypotheses for how ToxR and TcpP function. Along with determining the structure of ToxR and TcpP, of interest also is overexpression, purification and crystallization of the structures of membrane effector proteins required for the activity of each: ToxS in the case of ToxR, and TcpH in the case of TcpP. The specific aims of the proposal are as follows: (i.) To purify a 6-His tagged version of full length ToxR for crystallization and structural determination with and without its toxT promoter binding site or its binding site in the ompU promoter (a promoter activated by ToxR independently of TcpP) (ii.) To purify a 6-His tagged version of full length TcpP for crystallization and structural determination with and without its toxT promoter-binding site (iii.) To obtain structural data on co-crystals of ToxR and TcpP on the toxT promoter (to determine whether protein-protein interactions affect DNA binding) (iv.) To purify FLAG epitope- tagged versions of ToxS and TcpH for crystallization and structural determination alone or in a co-crystal with ToxR or TcpP

描述（由申请人提供）：该提案旨在开发从霍乱弧菌（人类霍乱疾病的病原体）中过度表达、纯化和结晶膜定位转录控制蛋白的方法。 ToxR 和 TcpP 是具有细胞质 DNA 结合/激活结构域的双主题膜蛋白。它们通过激活toxT表达的能力来控制重要毒力因子的表达，toxT是编码霍乱毒素和毒素共调节菌毛的基因的激活剂。 ToxR 和 TcpP 的 DNA 结合/激活结构域与翼状螺旋-转-螺旋 (w-HTH) 蛋白家族同源。其他 w-HTH 结构域的结构已得到解决，但 ToxR 和 TcpP 不寻常的膜拓扑结构，以及旨在确定它们如何识别 DNA 和激活 toxT 转录的重要初步数据，迫使人们对解决它们与操纵基因 DNA 结合时的晶体结构产生兴趣。完成后，这项研究将使我们能够区分 ToxR 和 TcpP 如何发挥作用的不同假设。除了确定 ToxR 和 TcpP 的结构外，我们还感兴趣的是每种活性所需的膜效应蛋白结构的过表达、纯化和结晶：对于 ToxR 来说是 ToxS，对于 TcpP 来说是 TcpH。 该提案的具体目标如下： (i.) 纯化全长 ToxR 的 6-His 标记版本，用于结晶和结构测定，有或没有其 toxT 启动子结合位点或其 ompU 启动子中的结合位点（ToxR 独立于 TcpP 激活的启动子） (ii.) 纯化全长 TcpP 的 6-His 标记版本，用于有或没有其 toxT 的结晶和结构测定 (iii.) 获得 toxT 启动子上 ToxR 和 TcpP 共晶的结构数据（以确定蛋白质-蛋白质相互作用是否影响 DNA 结合） (iv.) 纯化 ToxS 和 TcpH 的 FLAG 表位标记版本，用于单独结晶和结构测定或与 ToxR 或 TcpP 共晶