Structural Analysis of Vibrio cholerae Virulence Gene Regulatory Proteins

霍乱弧菌毒力基因调控蛋白的结构分析

• 批准号: 7189792
• 负责人: Fredrick Jon Kull
• 金额: $ 39.98万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-12-15 至 2011-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7189792
• 关键词: Affinity; Amino Acids; Bacterial Infections; Binding; Binding Sites; C-terminal; Cell Density; Chitin; Cholera; Cholera Toxin; Complex; Crystallization; DNA; DNA Binding; DNA Binding Domain; Development; Dimerization; Disease; Epidemic; Family; Gene Expression; Gene Expression Regulation; Genes; Goals; Laboratories; Lead; Length; Light; Molecular; Mutagenesis; Numbers; Pathogenesis; Pathogenicity; Pharmaceutical Preparations; Pilum; Proteins; Regulation; Research Personnel; Resolution; Signal Transduction; Site-Directed Mutagenesis; Stimulus; Structure; System; Testing; Toxin; Transcription Coactivator; Transcriptional Activation; Vibrio cholerae; Virulence; Virulence Factors; Winged Helix; Work; base; environmental change; genetic regulatory protein; inhibitor/antagonist; insight; intein; member; novel; prevent; programs; promoter; protein function; quorum sensing; response; small molecule; structural biology; transcription factor

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. 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 cognate promoters to control gene expression and, ultimately, how they are influenced by environmental stimuli. Through a collaborative effort of laboratories with expertise in structural biology, virulence gene regulation and pathogenesis, we have obtained the crystal structure of AphA at 2.2 A resolution. Its structure reveals the presence of a winged-helix DNA binding domain and a topologically unique dimerization domain. Aim 1 focuses on obtaining high resolution structures of (1) AphA with its cognate binding site, (2) AphB alone and in the presence of its cognate binding site, and (3) AphA and AphB together in a ternary complex with DNA. In addition, specific structural predictions will be tested using site-directed mutagenesis. Aims 2 and 3 focus on obtaining high resolution structures of ToxT and HapR in the absence and presence of their binding sites and mutagenesis will also be carried out to test structural predictions. This proposed work will significantly increase our understanding of how these proteins regulate virulence gene expression in order to facilitate efforts to identify new molecules that interfere with their functions and which may serve as novel antivirulence drugs.

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）的表达通过涉及几种激活蛋白的转录级联发生，并作为细菌毒力调节的范例。 AphA 和 AphB 通过 tcpPH 启动子处的新型相互作用启动级联的表达。 AphA 是新调节家族的成员，AphB 是 LysR 型激活剂，是最大的转录调节家族之一。一旦表达，TcpP/TcpH 与同源跨膜激活剂 ToxR/ToxS 之间的合作会激活 toxT 启动子。 ToxT 是一种 AraC 型调节因子，然后直接激活初级毒力因子的启动子。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。其中一种刺激物，细胞密度，通过群体感应系统调节器 HapR 影响毒力级联，HapR 抑制 aphA 启动子的表达。该提案的长期目标是了解毒力基因调控的分子基础，以促进制定更好的策略来预防和治疗细菌性疾病。实现这些目标需要了解特定的调节蛋白如何在其同源启动子上发挥作用以控制基因表达，以及最终它们如何受到环境刺激的影响。通过具有结构生物学、毒力基因调控和发病机制专业知识的实验室的合作努力，我们获得了分辨率为 2.2 A 的 AphA 晶体结构。其结构揭示了翼状螺旋 DNA 结合域和拓扑独特的二聚化域的存在。目标 1 侧重于获得 (1) AphA 及其同源结合位点，(2) 单独的 AphB 及其同源结合位点，以及 (3) AphA 和 AphB 与 DNA 形成三元复合物的高分辨率结构。此外，将使用定点诱变来测试特定的结构预测。目标 2 和 3 重点是在存在和不存在结合位点的情况下获得 ToxT 和 HapR 的高分辨率结构，并且还将进行诱变以测试结构预测。这项拟议的工作将显着增加我们对这些蛋白质如何调节毒力基因表达的理解，以促进识别干扰其功能并可作为新型抗毒力药物的新分子。