Structure and Function of Response Regulator Proteins

描述(申请人提供)：很大一部分细菌信号是由“双组分”系统介导的，该系统利用传感器组氨酸蛋白激酶和反应调节蛋白之间的磷酸转移的保守机制。许多双组分系统存在于单个细菌中，并经常在对致病过程重要或必不可少的途径中发挥作用。动物体内不存在双组分蛋白质，这使得它们成为寻找新的抗菌剂的诱人目标。反应调节子的保守结构域起着磷酸化激活开关的作用，控制效应结构域的活性和输出反应。这一应用侧重于反应调节蛋白激活机制的结构和生化表征。特别是，该项目试图确定调节结构域和效应结构域之间的分子内相互作用与作为转录因子的OmpR/Phob亚家族的功能二聚体中调节结构域之间的分子间相互作用对调节的相对贡献。这些研究将涉及嵌合反应调节蛋白的构建和表征，活性二聚体中相互作用表面的确定，以及X射线结晶学的结构表征。申请人的研究旨在详细描述少数具有代表性的反应调节因子的调控机制，期望不仅对了解这些单个蛋白质具有重要意义，而且对确定一个大的信号蛋白家族中的相似和不同之处具有重要意义。这个系统提供了一个机会来解决序列和结构相似性在多大程度上可以用来预测功能机制上的相似性，这是在这个蛋白质组学时代出现的一个重要问题。更多的研究集中在有助于受体介导的适应的细菌趋化系统的组件上，特别是对多域反应调节因子甲酯酶CHEB中的调控相互作用的研究，以及Ched和Check蛋白的结构特征的研究。

DESCRIPTION (provided by applicant): A large fraction of bacterial signaling is mediated by "two-component" systems that utilize a conserved mechanism of phosphotransfer between a sensor histidine protein kinase and a response regulator protein. Numerous two-component systems are present in a single bacterium and often function in pathways that are important or essential for pathogenesis. The absence of two-component proteins from animals has made them attractive targets for pursuit of new antimicrobial agents. The conserved domain of response regulators functions as a phosphorylation-activated switch, controlling the activity of the effector domain and the output response. This application focuses on structural and biochemical characterization of the mechanism of activation of response regulator proteins. In particular, the project seeks to determine the relative regulatory contributions of intramolecular interactions between the regulatory and effector domains versus intermolecular interactions between the regulatory domains within functional dimers of the large OmpR/PhoB subfamily of response regulators that function as transcription factors. The studies will involve construction and characterization of chimeric response regulator proteins, determination of surfaces of interaction within active dimers, and structural characterization by X-ray crystallography. The applicant's studies are aimed at providing a detailed description of the regulatory mechanisms of a small number of representative response regulators, with the expectation of significance not only to an understanding of these individual proteins, but also to establishing the similarities and differences within a large family of signaling proteins. This system provides an opportunity to address the extent to which sequence and structural similarity can be used to predict similarity in mechanism of function, an important question that is emerging in this era of proteomics. Additional studies are focused on components of bacterial chemotaxis systems that contribute to receptor-mediated adaptation, specifically studies of regulatory interactions in the multi-domain response regulator methylesterase CheB and structural characterization of proteins CheD and CheC.