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）：弧菌霍乱引起致命的流行腹泻病霍乱。其主要的毒力因子的表达，毒素可调节的pilus和霍乱毒素是通过涉及多种激活蛋白的转录级联反应发生的，并作为调节细菌毒力的范例。 APHA和APHB通过TCPPH启动子的一种新型相互作用启动了级联反应的表达。 APHA是一个新的监管家族的成员，APHB是LYSR型激活剂，这是最大的转录调节家庭之一。一旦表达，TCPP/TCPH与同源跨膜激活剂TOXR/TOXS之间的合作激活了TOXT启动子。 TOXT是ARAC型调节剂，然后直接激活主要毒力因子的启动子。这些启动子的转录激活仅是为了响应某些环境刺激而发生。一种这样的刺激，细胞密度，通过群体传感系统调节剂HAPR影响毒力级联反应，该级子抑制了APHA启动子的表达。该提案的长期目标是了解毒力基因调节的分子基础，以促进制定更好的预防和治愈细菌疾病的策略。实现这些目标需要了解特定的调节蛋白如何在其同源启动子中发挥作用以控制基因表达，并最终如何受环境刺激的影响。通过在结构生物学，毒力基因调节和发病机理方面具有专业知识的实验室的合作努力，我们在2.2 A分辨率下获得了APHA的晶体结构。它的结构揭示了有翼螺旋DNA结合结构域和拓扑独特的二聚体域的存在。 AIM 1专注于（1）APHA的高分辨率结构，其同源结合位点，（2）APHB，并且在其同源结合位点的存在下，以及（3）APHA和APHB在带有DNA的三元复合物中一起。此外，将使用定向诱变测试特定的结构预测。目的2和3的重点是在没有结合位点和诱变的情况下，在不存在和存在的情况下获得TOXT和HAPR的高分辨率结构，以测试结构预测。这项提出的工作将大大提高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能充当新型抗电力药物的新分子的努力。