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

描述（由申请人提供）：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。最近爆发的耐多药结核菌株以及结核病和艾滋病的致命协同作用强调需要新的和更有效的结核病治疗方法。我们提出表征PhoR传感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至关重要。全球基因谱研究表明，php - phor至少有44个基因受到正调控，70个基因受到负调控。因此，该信号系统必须在病原体适应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，没有关于这一双组分系统的结构数据，信号转导的机制也是未知的。本提案的具体目的是：(1)确定PhoP的晶体结构；(2)定义PhoP识别DMA序列的机制；(3)研究PhoR各结构域在二聚体形成和蛋白活性调控中的作用；(4)确定截断结构域和全长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端结构域并获得了全长PhoP的晶体；（ii）确定了几个结合PhoP的启动子，并绘制了PhoP结合位点，从中我们将确定用于PhoP- dna复合物结构测定的最佳DMA序列；（iii）纯化全长PhoR并制备用于产生各种截断PhoR结构域的表达构建体。我们将采用分而治之的策略来研究与全长蛋白平行的分离结构域。分离结构域的结构和功能信息将导致成功确定全长蛋白的结构。完整的PhoR结构将提供第一个完整的膜蛋白结构的一大组传感器组氨酸激酶。这项研究的结果将导致PhoP-PhoR信号转导的详细分子机制，从而更好地了解病原体如何适应细胞内环境。高分辨率晶体结构也将为设计更好的治疗方法提供基础。

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.