STRUCTURE AND FUNCTION OF RESPONSE REGULATOR PROTEINS

真核生物和原核生物对多种反应的调控 细胞涉及一组蛋白质家族，这些蛋白质发挥着分子开关的作用 启用和禁用特定效应器功能。非活动状态之间的切换 而活性状态是由诱导的构象变化引起的 通过蛋白质侧链的共价修饰或通过结合 小分子。在原核细胞中，一类反应调节因子 由同源开关结构域和特定效应器组成的蛋白质 域，调节对不断变化的环境条件的反应。这个 调节结构域通过天冬氨酸侧的磷酸化而打开 链，并通过本征的酰基磷酸酯的水解而关闭 磷酸酶活性。调节结构域的磷酸化通过 从相关的组氨酸蛋白激酶进行的磷酸转移。这些 磷酸转移介导的信号转导系统广泛存在 整个细菌王国，并调节过程，如细胞 运动、分化、运输、新陈代谢和建立 寄主/病原体的相互作用。拟议的研究重点是 这些磷酸化激活开关的结构/功能分析 结构域，目的是了解它们的分子机制 行动。具体地说，磷转移和磷转移的机制是什么 磷酸盐水解法？这种构象变化的性质是什么？ 是由磷酸化诱导的吗？以及监管领域是如何传递的 它对效应器领域的影响？细菌趋化蛋白， Chey负责调节鞭毛的旋转，是 磷酸化激活的调节域。这128个氨基酸是单一的 结构域蛋白具有典型的α/β折叠，由中心5- 搁浅的平行贝塔薄片两侧都有阿尔法螺旋。这个 位于β-折叠的C-末端边缘的活性中心由 在细菌家族中高度保守的残基 应对监管机构。活性中心酸性口袋，由一簇 羧酸侧链，是磷酸化和二价金属的中心 离子结合。以CHEY为模型调控域，其机制 磷转移和去磷酸化将通过构建 特定部位的突变，表征了 这些改变蛋白的磷酸化/去磷酸化活性 使用小分子磷供体，并确定了其结构 相关突变蛋白的X射线结晶学分析。它的X射线结构 Chey的活性构象将通过筛选 稳定活性形式的突变，并通过使用小分子 能够使晶体中的酪氨酸氨基转移酶磷酸化的磷供体。作为一个步骤 向确定效应器功能调节机制的方向 开关结构域的磷酸化，结构分析两个额外的 结合甲酯酶(CHEB)和DNA结合的反应调节因子 (OmpR)效应域将被启动。

Regulation of a variety of responses in both eukaryotic and prokaryotic cells involves families of proteins that function as molecular switches to turn on and off particular effector functions. The switch between inactive and active states results from conformational changes that are induced either by covalent modifications of protein side chains or by binding of small molecules. In prokaryotic cells, a family of response regulator proteins composed of homologous switch domains and specific effector domains, mediate responses to changing environmental conditions. The regulatory domain is turned on by phosphorylation of an aspartate side chain and turned off by hydrolysis of the acyl phosphate by an intrinsic phosphatase activity. Phosphorylation of the regulatory domain occurs via phosphotransfer from an associated histidine protein kinase. These phosphotransfer mediated signal transduction systems are widespread throughout the bacterial kingdom and regulate processes such as cell motility, differentiation, transport, metabolism, and establishment of host/pathogen interactions. The proposed research focuses on structure/function analysis of these phosphorylation-activated switch domains with the goal of understanding the molecular mechanism of their action. Specifically, what are the mechanisms of phosphotransfer and phosphate hydrolysis? What is the nature of the conformational change that is induced by phosphorylation? And how does the regulatory domain transmit its effects to the effector domain? The bacterial chemotaxis protein, CheY, which regulates flagellar rotation, is representative of the phosphorylation-activated regulatory domains. This 128 amino acid single domain protein has a classic alpha/beta fold consisting of a central five- stranded parallel beta sheet flanked on both sides by alpha helices. The active site, located at the C-terminal edge of the beta sheet is composed of residues that are highly conserved among the family of bacterial response regulators. The active site acidic pocket, formed by a cluster of carboxylate side chains, is the site of phosphorylation and divalent metal ion binding. Using CheY as a model regulatory domain, the mechanisms of phosphotransfer and dephosphorylation will be addressed by constructing site specific mutations, characterizing the phosphorylating/dephosphorylating activities of these altered proteins using small molecule phospho-donors, and determining the structures of relevant mutant proteins by X-ray crystallography. The X-ray structure of the active conformation of CheY will be approached both by screening for mutations that stabilize the active form, and by using small molecule phospho-donors capable of phosphorylating CheY in crystals. As a step towards determining the mechanism of regulation of effector function via phosphorylation of the switch domain, structural analysis of two additional response regulators with attached methylesterase (CheB) and DNA binding (OmpR) effector domains will be initiated.