Structural Biology of Multifunctional Bacterial Phosphatases

描述（由申请人提供）：Rap蛋白包括细胞质蛋白的同源家族，通过显著不同的机制调节细菌基因表达。被称为Phr肽的分泌信号被输入细胞，在那里它们与Rap蛋白结合并抑制其活动。我们研究的总体目标是确定Rap-Phr信号系统如何机制地调节细菌信号转导。Rap蛋白的一个亚群通过增加Spo0F（孢子形成信号转导通路中的中心蛋白）催化调节性天冬氨酸去磷酸化的速率，对枯草芽孢杆菌的孢子形成起到负调控作用。Rap蛋白的另一个亚群通过抑制早期能力基因表达的主要转录调控因子ComA，从结合到靶DNA启动子，从而下调枯草芽孢杆菌遗传能力的发展。其他枯草芽孢杆菌Rap蛋白不是本提案的直接研究对象，它们调节遗传元件的移动性并拮抗除ComA以外的转录因子的活性。从公共卫生的角度来看，确定Rap蛋白如何调节细菌信号转导是很重要的，因为Rap蛋白调节致病生物的毒力表型。例如，炭疽杆菌是炭疽病的病原体，它的产孢受到编码在其染色体和毒力质粒pX01上的Rap蛋白的抑制。这种抑制是炭疽芽胞杆菌在被感染宿主中成为致病性营养细胞所必需的。Rap蛋白如何在机制上调节其靶蛋白的多种活性尚不清楚。有趣的是，Phr肽是由rap基因3端重叠的基因编码的小蛋白通过输出成熟途径产生的。成熟的Phr五肽分子被导入细胞，与Rap蛋白结合，抑制Rap蛋白对基因表达的负调控作用。在Aim 1中，我们将确定RapC如何通过抑制ComA与目标DNA启动子的结合来负性调节枯草芽孢杆菌的遗传能力，并展示分泌信号PhrC如何通过抑制RapC和ComA的相互作用来促进遗传能力。在Aim 2中，我们将揭示RapA如何通过增加Spo0F去磷酸化的速率来抑制枯草芽孢杆菌的产孢，并确定分泌信号PhrA如何通过抑制RapA和Spo0F的相互作用来诱导产孢。本文提出的x射线晶体学、生物化学和细菌遗传学研究将首次揭示Rap蛋白如何调节其靶蛋白的活性以及Phr肽如何抑制Rap蛋白的功能。揭示Rap-Phr功能的分子机制将使我们能够实现设计调节细菌信号转导的抗菌药物的长期目标。独特的细胞蛋白调节细菌的生长、增殖和毒力，包括常见的人类病原体。细菌感染变得越来越难以治疗，对公共卫生的威胁也越来越大，因为它们对现有的抗菌药物产生了耐药性。我们工作的长期目标是利用生物化学、生物物理和遗传方法来阐明细菌调节蛋白的功能，并设计针对这些蛋白的新型抗菌药物。

DESCRIPTION (provided by applicant): Rap proteins comprise a homologous family of cytoplasmic proteins that regulate bacterial gene expression via remarkably different mechanisms. Secreted signals, called Phr peptides, are imported into the cell where they bind to Rap proteins and repress their activities. The overall goal of our research is to determine how Rap-Phr signaling systems function mechanistically to regulate bacterial signal transduction. One subset of Rap proteins negatively regulates sporulation in B. subtilis by increasing the rate at which Spo0F, a central protein in the sporulation signal transduction pathway, catalyzes the dephosphorylation of a regulatory aspartic acid. Another subset of Rap proteins downregulates the development of genetic competence in B. subtilis by inhibiting ComA, the master transcriptional regulator of early competence gene expression, from binding to target DNA promoters. Additional B. subtilis Rap proteins that are not subjects of immediate study in this proposal regulate the mobility of genetic elements and antagonize the activity of transcription factors other than ComA. It is important from a public health standpoint to determine how Rap proteins regulate bacterial signal transduction because Rap proteins regulate virulence phenotypes in pathogenic organisms. For example, sporulation is repressed in Bacillus anthracis, the causative agent of the disease anthrax, by Rap proteins encoded on its chromosome and virulence plasmid, pX01. This repression is required for B. anthracis to become pathogenic vegetative cells in the infected host. How Rap proteins function mechanistically to regulate the diverse activities of their target proteins is not understood. Interestingly, Phr peptides are generated by an export maturation pathway from small proteins encoded by genes that overlap with the 3 end of the rap genes. Mature Phr pentapeptide molecules are imported into the cell where they bind to Rap proteins and inhibit their negative regulatory effects on gene expression. In Aim 1 we will determine how RapC negatively regulates genetic competence in B. subtilis by inhibiting the binding of ComA to target DNA promoters and also show how the secreted signal, PhrC, promotes genetic competence by inhibiting the interaction of RapC and ComA. In Aim 2 we will reveal how RapA inhibits B. subtilis sporulation by increasing the rate of Spo0F dephosphorylation and also determine how the secreted signal, PhrA, induces sporulation by inhibiting the interaction of RapA and Spo0F. The X-ray crystallographic, biochemical, and bacterial genetic studies proposed here will reveal, for the first time, how Rap proteins regulate the activities of their target proteins and how Phr peptides inhibit Rap protein function. Revealing the molecular mechanisms of Rap-Phr function will enable us to accomplish our long-term goal of designing antibacterial drugs that modulate bacterial signal transduction. PUBLIC HEALTH RELEVANCE Unique cellular proteins modulate the growth, proliferation, and virulence of bacteria, including common human pathogens. Bacterial infections are becoming increasingly difficult to treat, and an escalating threat to public health, as they acquire resistance to existing antibacterial drugs. The long-term goal of our work is to use biochemical, biophysical, and genetic approaches to elucidate the functions of bacterial regulatory proteins, and to design new classes of antibacterial drugs that target these proteins.