Structure and Function of Response Regulator Proteins

描述（由申请人提供）：细菌在人体健康中起着重要作用。复杂的细菌群落是正常生理的重要组成部分，通过对人类微生物群的研究，人们越来越了解这一点。其他细菌是众所周知的病原体，可以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有许多共同的基本特征。其中最重要的是细菌监测细胞内和细胞外环境的能力，并对变化的条件产生适当的适应性反应。将刺激与输出反应耦合的最普遍机制是一个“双组分系统”磷传递途径，涉及一个传感组氨酸蛋白激酶和一个产生输出反应的磷酸化激活反应调节因子。到目前为止，这种通用的管理方案已经在100万到20万个系统中得到了应用。虽然核心特征，特别是组氨酸激酶和反应调节因子的单个结构域的结构和生化活性是保守的，但在如何配置它们以优化单个系统的特定需求方面存在很大差异。二十年来的研究主要集中在体外双组分蛋白的生化表征上，揭示了不同系统中同源蛋白的自磷酸化、磷酸酶、磷酸化转移和自去磷酸化活性在量级上存在很大差异。由于无法在体内直接测量不稳定蛋白磷酸化，对这些参数与细胞内功能的关系的理解受到了阻碍。该项目将利用最近开发的方法，使这些测量能够建立蛋白质水平，酶活性和途径结构配置的基本原则，以优化适应性和行为。现有的方法将扩展到反应调节因子磷酸化的动力学表征，允许首次确定细胞内组氨酸激酶的对立激酶和磷酸酶活性的速率，以及这些速率如何被刺激改变。先前的研究表明，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滞后于所有其他双组分蛋白的保守活性。本项目将通过结合结构和生化方法来定义两大类组氨酸激酶HisKA和HisKA_3中磷酸酶活性的酶促机制来解决这一问题。总之，拟议的研究将填补当前知识的关键空白，为研究双组分信号系统配置提供新方法，确定先前建立的体外参数与体内系统行为的关系，并定义具有预测能力的基本原理，以理解许多尚未探索的双组分系统。这种微生物调控的基本知识对宿主-微生物相互作用至关重要，将为益生菌和抗生素治疗策略的发展提供坚实的基础。

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.