Structural Analysis of Vibrio cholerae Virulence Gene Regulatory Proteins

描述（由申请人提供）：霍乱弧菌引起致命的流行性腹泻病霍乱。其主要毒力因子，毒素共调节菌毛和霍乱毒素的表达，发生通过涉及几个激活蛋白的转录级联反应，并作为细菌毒力的调节范例。AphA和AphB通过在tcpPH启动子处的新型相互作用启动级联的表达。AphA是一个新的调节因子家族的成员，AphB是一个LysR型激活因子，是最大的转录调节因子家族之一。一旦表达，TcpP/TcpH和同源跨膜激活因子ToxR/ToxS之间的合作激活toxT启动子。ToxT是一种AraC型调节因子，然后以脂肪酸依赖性方式直接激活主要毒力因子的启动子。这些不同启动子的转录激活仅在响应某些环境刺激时发生。一种这样的刺激，细胞密度，通过群体感应系统调节剂HapR抑制aphA启动子的表达来影响毒力级联。本项目的长期目标是了解致病基因调控的分子基础，以促进开发更好的预防和治疗细菌性疾病的策略。实现这些目标需要了解特定的调节蛋白如何在其启动子中发挥作用以控制基因表达，以及最终如何受环境刺激的影响。通过与具有结构生物学、毒力基因调控和发病机理专业知识的实验室的合作，我们已经解决了AphA、AphB、HapR和ToxT的晶体结构。该建议将建立在这些结构数据以及我们的功能结果的基础上，以便继续阐明霍乱弧菌毒力基因调控所需的详细机制。在目的1中，我们建议确定AphA，AphB和HapR与其各自的DNA结合位点复合的晶体结构，使我们能够观察DNA结合后发生的结构变化。在目标2中，我们计划表征AphB和HapR的配体结合口袋。由于这些蛋白质的天然配体是未知的，我们计划识别HapR和AphB的小分子配体，然后可视化通过配体结合在蛋白质中诱导的结构变化。目的3研究脂肪酸结合对霍乱弧菌毒力基因表达的主要调控因子ToxT活性的调节机制。除了晶体学，我们将利用基于结构的定点诱变，生物化学活性测定，生物物理表征测定，结合和构象变化的光谱表征，以及基于计算和NMR的方法来识别配体。这些研究将极大地阐明参与细菌毒力基因表达调控的蛋白质的机制和结构作用。 这些知识将有助于识别干扰调节级联反应的分子，并可能导致新疗法的开发。

DESCRIPTION (provided by applicant): Vibrio cholerae causes the fatal epidemic diarrheal disease cholera. The expression of its primary virulence factors, toxin-coregulated pilus and cholera toxin, occurs via a transcriptional cascade involving several activator proteins and serves as a paradigm for the regulation of bacterial virulence. AphA and AphB initiate the expression of the cascade by a novel interaction at the tcpPH promoter. AphA is a member of a new regulator family and AphB is a LysR-type activator, one of the largest transcriptional regulatory families. Once expressed, cooperation between TcpP/TcpH and the homologous transmembrane activators ToxR/ToxS activates the toxT promoter. ToxT, an AraC-type regulator, then directly activates the promoters of the primary virulence factors in a fatty acid dependent manner. Transcriptional activation at these various promoters occurs only in response to certain environmental stimuli. One such stimulus, cell density, influences the virulence cascade through the quorum sensing system regulator HapR which represses the expression of the aphA promoter. The long term goals of this proposal are to understand the molecular basis of virulence gene regulation so as to facilitate the development of better strategies to prevent and cure bacterial diseases. Achieving these goals requires an understanding of how the specific regulatory proteins function at their promoters to control gene expression and, ultimately, how they are influenced by environmental stimuli. Through a collaborative effort involving laboratories with expertise in structural biology, virulence gene regulation, and pathogenesis, we have solved crystal structures of AphA, AphB, HapR, and ToxT. This proposal will build upon this structural data, as well as our functional results, in ordr to continue to elucidate the detailed mechanisms required for regulation of the V. cholerae virulence genes. In Aim 1, we propose to determine the crystal structures of AphA, AphB, and HapR in complex with their respective DNA binding sites, allowing us to observe the structural changes that take place upon DNA binding. In Aim 2, we plan to characterize the ligand binding pockets of AphB and HapR. As the natural ligands for these proteins are not known, we plan to identify small molecule ligands for HapR and AphB, and then visualize the structural changes induced in the proteins by ligand binding. Aim 3 carries on our investigation of the mechanism by which fatty acid binding regulates the activity of ToxT, the master regulator of virulence gene expression in V. cholerae. In addition to crystallography, we will utilize structure based site directed mutagenesis, biochemical activity assays, biophysical characterization assays, spectroscopic characterization of binding and conformational change, and computational and NMR based methods for identifying ligands. These studies will greatly clarify the mechanistic and structural roles of proteins involved in the regulation of bacterial virulence gene expression. Such knowledge will facilitate the identification of molecules interfering with regulatory cascades, and could lead to the development of novel therapeutics.