Structural Analysis of Vibrio cholerae Virulence Gene Regulatory Proteins

描述(申请人提供)：霍乱弧菌导致致命的流行性腹泻疾病霍乱。其主要毒力因子毒素共调节的菌毛和霍乱毒素的表达通过涉及几个激活蛋白的转录级联发生，并作为调节细菌毒力的范例。APHA和AphB通过在tcpPH启动子上的一种新的相互作用启动级联蛋白的表达。APHA是一个新的调控家族的成员，AphB是一个LysR类型的激活物，是最大的转录调控家族之一。一旦表达，TCPP/TcpH和同源跨膜激活剂ToxR/ToxS之间的合作就激活了oxT启动子。ToxT是一种AraC型调节因子，然后以脂肪酸依赖的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只发生在对某些环境刺激的反应中。其中一个这样的刺激，细胞密度，通过群体感应系统调节因子HapR影响毒力级联反应，抑制APHA启动子的表达。这项建议的长期目标是了解毒力基因调控的分子基础，以促进开发更好的预防和治疗细菌疾病的策略。要实现这些目标，需要了解特定的调控蛋白在启动子上如何发挥作用来控制基因表达，并最终了解它们是如何受到环境刺激的影响的。通过在结构生物学、毒力基因调控和发病机制方面的专业实验室的共同努力，我们已经解决了APHA、AphB、HapR和ToxT的晶体结构。这项建议将建立在这些结构数据的基础上，以及我们在ORDR中的功能结果，以继续阐明霍乱弧菌毒力基因调控所需的详细机制。在目标1中，我们建议确定APHA、AphB和HapR的晶体结构与它们各自的DNA结合部位，使我们能够观察DNA结合时发生的结构变化。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尚不清楚，我们计划确定HapR和AphB的小分子配体，然后可视化配体结合引起的蛋白质结构变化。目的研究脂肪酸结合调节霍乱弧菌毒力基因表达的主要调控因子ToxT活性的机制。除了结晶学，我们还将利用基于结构的定点突变、生化活性分析、生物物理表征、结合和构象变化的光谱表征，以及基于计算和核磁共振的方法来识别配体。这些研究将极大地阐明参与调节细菌毒力基因表达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发展。

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.