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: 9025669
• 负责人: Fredrick Jon Kull
• 金额: $ 46.73万
• 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 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9025669
• 关键词: Affect; Affinity; Bacterial Infections; Binding; Binding Proteins; Binding Sites; Biochemical; Biological Assay; Carbon; Cell Density; Cessation of life; Characteristics; Cholera; Cholera Toxin; Collaborations; Complex; Crystallography; DNA; DNA Binding; DNA Sequence; Data; Development; Dimerization; Disease; Docking; Enteral; Epidemic; Family; Family member; Fatty Acids; Foundations; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Goals; Homologous Gene; Homology Modeling; Indium; Investigation; Knowledge; Laboratories; Lead; Ligand Binding; Ligands; Methods; Molecular; Monounsaturated Fatty Acids; Mutation; Nature; Operon; Pathogenesis; Pathogenicity; Pharmaceutical Preparations; Pilum; Process; Proteins; Regulation; Research Design; Resolution; Role; Site-Directed Mutagenesis; Staging; Stimulus; Structure; Structure-Activity Relationship; System; Toxin; Transcriptional Activation; Vibrio cholerae; Virulence; Virulence Factors; World Health Organization; base; biophysical properties; chemical genetics; dimer; disorder control; genetic approach; genetic regulatory protein; improved; insight; member; mutant; novel; novel therapeutics; palmitoleic acid; pathogenic bacteria; prevent; promoter; protein function; quorum sensing; response; small molecule; small molecule inhibitor; structural biology; therapeutic development

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.

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。通过涉及实验室结构生物学，毒力基因调节和发病机理方面的实验室的协作努力，我们解决了APHA，APHB，HAPR和TOXT的晶体结构。该建议将基于此结构数据以及我们的功能结果，以继续阐明调节V.霍乱毒力性基因所需的详细机制。在AIM 1中，我们建议确定APHA，APHB和HAPR的晶体结构与它们各自的DNA结合位点复杂，从而使我们能够观察到DNA结合时发生的结构变化。在AIM 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，因此我们计划鉴定HAPR和APHB的小分子配体，然后可视化配体结合蛋白质诱导的结构变化。 AIM 3对我们研究了脂肪酸结合来调节Toxt的活性的机制，Toxt是霍乱弧菌中毒力基因表达的主要调节剂的活性。除晶体学外，我们还将利用基于结构的位点定向诱变，生化活性测定，生物物理表征分析，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。这些研究将大大阐明与细菌毒力基因表达相关的蛋白质的机械和结构作用。 这种知识将促进鉴定分子干扰调节级联反应，并可能导致新型治疗学的发展。

项目成果

An Enantiodefined Conformationally Constrained Fatty Acid Mimetic and Potent Inhibitor of ToxT.

一种对映体构象受限的脂肪酸模拟物和 ToxT 的有效抑制剂。

• DOI: 10.1021/acsmedchemlett.1c00378
• 发表时间: 2021
• 期刊: ACS medicinal chemistry letters
• 影响因子: 4.2
• 作者: Markham,LaurenE;Tolbert,JessicaD;Kull,FJon;Midgett,CharlesR;Micalizio,GlennC
• 通讯作者: Micalizio,GlennC

A new class of inhibitors of the AraC family virulence regulator Vibrio cholerae ToxT.

AraC 家族毒力调节剂霍乱弧菌 ToxT 的一类新抑制剂。

• DOI: 10.1038/srep45011
• 发表时间: 2017
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Woodbrey,AnneK;Onyango,EvansO;Pellegrini,Maria;Kovacikova,Gabriela;Taylor,RonaldK;Gribble,GordonW;Kull,FJon
• 通讯作者: Kull,FJon