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: 7473212
• 负责人: SHUISHU WANG
• 金额: $ 4.08万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 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 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7473212
• 关键词: Acquired Immunodeficiency Syndrome; Antitubercular Agents; Bacteria; Behavior; Binding; Binding Sites; Biological; Biological Assay; C-terminal; Cell membrane; Cells; Complex; Condition; Crystallization; DNA; DNA Binding; DNA Binding Domain; DNA Sequence; Data; Dimerization; Disease Outbreaks; Elements; Environment; Gene Expression; Gene Expression Regulation; Genes; Growth; Helix (Snails); 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; ear helix; 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; tuberculosis treatment

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。最近爆发的多重耐药结核病菌株以及结核病和艾滋病的致命协同作用强调了对结核病新的和更有效的治疗的需要。我们建议表征的PhoR传感器组氨酸激酶和其同源的反应调节剂PhoP从结核分枝杆菌的信号转导机制。这种双组分系统对于Mtb的毒力和细胞内生长是必需的。全球基因谱研究表明，至少有44个基因是正调控和70个基因是负调控的PhoP-PhoR。因此，该信号系统必须在病原体适应其细胞内环境中发挥重要作用，使PhoP-PhoR系统成为新型抗结核药物的潜在靶点。然而，没有结构数据可用于这种双组分系统，信号转导的机制是未知的。本计划的具体目标是：（1）确定PhoP的晶体结构;（2）确定PhoP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;（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（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）鉴定了几种结合PhoP的启动子，并绘制了PhoP结合位点，从中我们将确定用于确定PhoP-DNA复合物结构的最佳DMA序列;和（iii）纯化的全长PhoR和制备的用于产生各种截短的PhoR结构域的表达构建体。我们将使用分而治之的策略来研究与全长蛋白质平行的孤立结构域。分离结构域的结构和功能信息将导致成功地确定全长蛋白质的结构。完整的PhoR的结构将提供一大群传感器组氨酸激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PhoP-PhoR信号转导的分子机制，从而更好地了解病原体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治疗方法提供基础。