Structural and functional studies of a PhoP-PhoR two-component system

PhoP-PhoR 双组分系统的结构和功能研究

• 批准号: 7626346
• 负责人: SHUISHU WANG
• 金额: $ 28.9万
• 依托单位: HENRY M. JACKSON FDN FOR THE ADV MIL/MED
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-20 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7626346
• 关键词: Acquired Immunodeficiency Syndrome; Antitubercular Agents; Bacteria; Behavior; Binding; Binding Sites; Biological; Biological Assay; C-terminal; Cell membrane; Cells; Complex; Crystallization; DNA; DNA Binding; DNA Binding Domain; DNA Sequence; Data; Dimerization; Disease Outbreaks; Elements; Environment; Gene Expression; Gene Expression Regulation; Genes; Growth; Individual; Integral Membrane Protein; Lead; Length; Maps; Mediating; Membrane; Membrane Proteins; Molecular; Molecular Conformation; Multi-Drug Resistance; Mutation; Mycobacterium tuberculosis; N-terminal; Pathogenesis; Phosphorylation; Phosphotransferases; Play; Population; Positioning Attribute; Proteins; Public Health; Regulation; Research; Research Personnel; Resolution; Role; Sequence Analysis; Signal Transduction; Signaling Protein; Specificity; Stress; Structure; System; Techniques; Therapeutic; Transcriptional Regulation; Tuberculosis; Virulence; Work; base; design; dimer; effective therapy; enzyme activity; in vivo; inhibitor/antagonist; killings; novel; pathogen; promoter; protein structure; protein-histidine kinase; response; sensor; sensor histidine kinase; small molecule; success; tuberculosis drugs

DESCRIPTION (provided by applicant): Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB), which kills 2 million people per year and infects nearly one-third of the world's population. Recent outbreaks of multi-drug resistant Mtb strains and the deadly synergy of TB and AIDS stress the need for new and more effective treatments for TB. We propose to characterize the signal transduction mechanism of the PhoR sensor histidine kinase and its cognate response regulator PhoP from Mtb. This two-component system is essential for virulence and intracellular growth of Mtb. Global gene profiling studies indicate that at least 44 genes are positively regulated and 70 genes are negatively regulated by PhoP-PhoR. Therefore, this signaling system must play an important role in adaptation of the pathogen to its intracellular environments, making the PhoP-PhoR system a potential target for novel anti-tuberculosis drugs. However, no structural data is available for this two-component system, and the mechanism of signal transduction is unknown. The specific aims of this proposal are (1) to determine the crystal structure of PhoP; (2) to define the mechanism of DMA sequence recognition by PhoP; (3) to investigate the role of each domain of PhoR in dimer formation and regulation of the protein activity; and (4) to determine crystal structures of truncated domains and the full-length PhoR protein. Toward achieving these aims, we have (i) determined the structure of the C-terminal domain of PhoP and obtained crystals of full-length PhoP; (ii) identified several promoters that bind PhoP and mapped the PhoP binding sites, from which we will identify optimal DMA sequences for structural determination of PhoP-DNA complexes; and (iii) purified full-length PhoR and prepared expression constructs for producing various truncated domains of PhoR. We will use a divide-and-conquer strategy to study the isolated domains in parallel with the full-length proteins. Structural and functional information of separate domains will lead to success in determining the structure of the full-length protein. The structure of intact PhoR will provide the first integral membrane protein structure of a large group of sensor histidine kinases. Results from this research will lead to a detailed molecular mechanism of signal transduction by PhoP-PhoR and thus a better understanding of how the pathogen adapts to intracellular environments. High resolution crystal structures will also provide a basis for the design of better therapeutics.

描述(申请人提供)：结核分枝杆菌(Mtb)是结核病(TB)的病原体，每年导致200万人死亡，感染世界近三分之一的人口。最近多药耐药结核分枝杆菌菌株的暴发以及结核病和艾滋病的致命协同作用突出了结核病新的和更有效的治疗方法的必要性。我们建议从Mtb中研究磷酸感受器组氨酸激酶及其同源反应调节因子Phop的信号转导机制。这种双组分系统对结核分枝杆菌的毒力和细胞内生长是必不可少的。全球基因图谱研究表明，至少有44个基因受到磷农药的正调控，70个基因受到磷农药的负调控。因此，该信号系统必须在病原体适应其细胞内环境方面发挥重要作用，使其成为新型抗结核药物的潜在靶点。然而，目前还没有关于这一双组分体系的结构数据，其信号转导机制也是未知的。本建议的具体目的是：(1)确定PhOP的晶体结构；(2)确定PhP识别DMA序列的机制；(3)研究Phor的每个结构域在二聚体形成和蛋白质活性调节中的作用；以及(4)确定截短结构域和全长Phor蛋白的晶体结构。为了实现这些目标，我们已经(I)确定了Phop的C-末端结构域的结构并获得了全长Phop的晶体；(Ii)鉴定了几个与Phop结合的启动子并定位了Phop的结合位点，从中我们将确定用于确定Phop-DNA复合体结构的最佳DMA序列；以及(Iii)纯化全长Phor并制备用于产生各种Phor截短结构域的表达构建体。我们将使用分而治之的策略在研究全长蛋白质的同时研究分离的结构域。不同结构域的结构和功能信息将导致成功确定全长蛋白质的结构。完整的phor结构将提供一大组组氨酸敏感酶的第一个完整的膜蛋白结构。这项研究的结果将导致详细的分子机制的信号转导，从而更好地了解病原菌如何适应细胞内环境。高分辨率的晶体结构也将为设计更好的疗法提供基础。