Structure and Function of Response Regulator Proteins

描述（由申请人提供）：细菌在人类健康中起着重要作用。复杂的细菌群落是正常生理学的重要组成部分，通过对人类微生物群的研究，人们越来越多地了解到这一点。其他细菌是众所周知的病原体，可导致危及生命的感染。无论是影响健康还是疾病，微生物与宿主的相互作用都具有许多基本特征。其中最重要的是细菌监测其细胞内和细胞外环境并对变化的条件引起适当的适应性反应的能力。将刺激与输出反应偶联的最普遍的机制是“双组分系统”磷酸转移途径，其涉及传感器组氨酸蛋白激酶和产生输出反应的磷酸化激活的反应调节剂。迄今为止，这种通用的调节方案已在超过200，000个系统中得到利用。虽然核心特征，特别是组氨酸激酶和反应调节剂的各个结构域的结构和生化活性是保守的，但它们如何配置以优化各个系统的特定需求存在很大差异。二十年的研究集中在体外双组分蛋白质的生物化学表征上，揭示了不同系统中同源蛋白质之间在幅度上差异很大的自磷酸化、磷酸酶、磷酸转移和自去磷酸化活性。由于无法直接测量体内不稳定蛋白质磷酸化，因此无法理解这些参数与细胞内功能的关系。该项目将利用最近开发的方法，使这些测量建立蛋白质水平，酶活性和途径结构如何配置的基本原则，以优化健身和行为。现有的方法将被扩展到动力学特性的反应调节磷酸化允许，第一次，确定的速率的对立的激酶和磷酸酶活性的组氨酸激酶在细胞内，以及如何改变这些利率的刺激。以往的研究表明，系统输出高度依赖于组氨酸激酶的磷酸酶活性。然而，这种活性及其酶促机制的表征落后于双组分蛋白质的所有其他保守活性。本研究将结合结构和生物化学的方法来研究两类主要的组氨酸激酶HisKA和HisKA_3中磷酸酶活性的酶机制。总之，拟议的研究将填补目前知识的关键空白，使新的方法来研究双组分信号系统的配置，确定以前建立的体外参数如何与系统在体内的行为，并定义基本原则与预测能力，以了解许多尚未探索的双组分系统。这种对宿主-微生物相互作用至关重要的微生物调节的基本知识将为益生菌和抗生素治疗策略的开发提供坚实的基础。

DESCRIPTION (provided by applicant): Bacteria play important roles in human health. Complex bacterial communities are important components of normal physiology, as is increasingly being understood through investigations of human microbiota. Other bacteria are well known as pathogens that can cause life-threatening infections. Whether impacting health or disease, interactions of microbes with hosts share many fundamental features. Paramount among these is the ability of bacteria to monitor their intracellular and extracellular environment and to elicit appropriate adaptive responses to changing conditions. The most prevalent mechanism for coupling stimuli to output responses is a "two-component system" phosphotransfer pathway involving a sensor histidine protein kinase and a phosphorylation-activated response regulator that generates the output response. This versatile regulatory scheme has been exploited in >200,000 systems identified to date. While core features, specifically, the structures and biochemical activities of individual domains of histidine kinases and response regulators, are conserved, there is great variation in how they are configured to optimize specific needs of individual systems. Two decades of research have focused on biochemical characterization of two-component proteins in vitro, revealing autophosphorylation, phosphatase, phosphotransfer, and autodephosphorylation activities that differ greatly in magnitude among homologous proteins in different systems. An understanding of how these parameters relate to function within cells has been hindered by an inability to directly measure labile protein phosphorylation in vivo. This project will utilize recently developed methodologies that enable such measurements to establish fundamental principles of how protein levels, enzyme activities, and pathway architecture are configured to optimize both fitness and behavior. The existing methodologies will be extended to kinetic characterization of response regulator phosphorylation allowing, for the first time, determination of the rates of the opposing kinase and phosphatase activities of histidine kinases within cells, and how these rates are altered by stimuli. Previous studies have shown that system output is highly dependent on the phosphatase activity of histidine kinases. However, characterization of this activity and its enzymatic mechanism lags behind all other conserved activities of two-component proteins. This project will address this issue by using combined structural and biochemical approaches to define the enzymatic mechanisms of phosphatase activity in the two major classes of histidine kinases, HisKA and HisKA_3. Together, the proposed studies will fill critical gaps in current knowledge, enable new approaches for investigating two-component signaling system configuration, determine how previously established in vitro parameters relate to system behavior in vivo, and define fundamental principles with predictive capabilities for understanding many as yet unexplored two-component systems. This fundamental knowledge of microbial regulation that is essential to host-microbe interactions will provide a firm underpinning for development of both probiotic and antibiotic therapeutic strategies.