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

描述(由申请人提供)：这项提案寻求开发从霍乱弧菌(人类霍乱病原体)过度表达、纯化和结晶膜定位转录控制蛋白的方法。ToxR和TCPP是具有胞质DNA结合/激活结构域的双主题膜蛋白。它们通过激活毒素T的表达来控制重要毒力因子的表达，毒素T是霍乱毒素编码基因和毒素共调控菌毛的激活因子。ToxR和TCPP的DNA结合/激活结构域与有翼螺旋-转角-螺旋(w-HTH)家族同源。其他w-HTH结构域的结构已经解决，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并激活toxT转录的重要初步数据，迫使人们对解决它们与操纵子DNA结合时的晶体结构感兴趣。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说。除了确定ToxR和TCPP的结构外，还需要研究ToxR和TCPP活性所需的膜效应蛋白结构的过表达、纯化和结晶：ToxR的ToxS和TCPP的TcpH。 该建议的具体目标如下：(一)提纯全长ToxR的6-His标记版本，用于结晶和结构测定，并结合和不结合其toxT启动子或其在ompU启动子(由ToxR独立于TCPP激活的启动子)中的结合位点(ii.)提纯全长TCPP的6-His标记版本，用于结晶和结构测定，并结合和不结合其毒素T启动子结合位点(III.)获得ToxT启动子上ToxR和TCPP共晶体的结构数据(确定蛋白质-蛋白质相互作用是否影响DNA结合)(iv.)提纯ToxS和TcpH的FLAG表位标记形式，用于单独或与ToxR或TCPP共晶结晶和结构测定

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