Structure and Function of Response Regulator Proteins

描述（由申请人提供）：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传递的保守机制。在单个细菌中存在许多双组分系统，并且通常在对发病机制重要或必要的途径中起作用。由于动物体内缺乏双组分蛋白，这使得它们成为研究新型抗菌剂的有吸引力的目标。反应调控的保守域作为磷酸化激活的开关，控制效应域的活性和输出响应。本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，该项目旨在确定作为转录因子的反应调节因子的大OmpR/PhoB亚家族的功能二聚体中调节域和效应域之间的分子内相互作用与调节域之间的分子间相互作用的相对调节贡献。这些研究将涉及嵌合反应调节蛋白的构建和表征，活性二聚体相互作用表面的测定，以及x射线晶体学的结构表征。申请人的研究旨在提供少量代表性反应调节因子的调控机制的详细描述，期望不仅对这些单个蛋白质的理解具有重要意义，而且对建立一个大的信号蛋白家族中的相似性和差异性具有重要意义。该系统提供了一个机会来解决序列和结构相似性可用于预测功能机制相似性的程度，这是这个蛋白质组学时代出现的一个重要问题。其他的研究集中在细菌趋化系统的组成部分，这些组成部分有助于受体介导的适应，特别是多结构域反应调节剂甲基酯酶CheB的调节相互作用和蛋白质CheD和CheC的结构表征的研究。

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.