STRUCTURE AND FUNCTION OF RESPONSE REGULATOR PROTEINS

真核生物和原核生物中多种反应的调控 细胞涉及蛋白质家族，这些蛋白质家族充当分子开关， 打开和关闭特定的效应器功能。 在非活动状态和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，反应调节因子家族 由同源开关结构域和特异性效应子组成的蛋白质 域，介导对不断变化的环境条件的反应。 的 调节结构域通过天冬氨酸侧的磷酸化而开启 链和关闭的水解的酰基磷酸的内在 磷酸酶活性 调节结构域的磷酸化通过以下途径发生： 从相关的组氨酸蛋白激酶磷酸转移。 这些 磷酸转移介导的信号转导系统是广泛存在的 在细菌界和调节过程，如细胞 运动，分化，运输，代谢和建立 宿主/病原体相互作用 拟议的研究重点是 这些磷酸化激活开关结构/功能分析 目的是了解其分子机制， 行动上 具体来说，磷酸转移和 磷酸盐水解？ 什么是构象变化的本质， 是由磷酸化引起的吗 监管领域如何传递 对效应域的影响 细菌趋化蛋白， CheY，调节鞭毛旋转，代表了 磷酸化激活的调节结构域。 这128个氨基酸单 结构域蛋白具有典型的α/β折叠，其由中心五个- 双股平行β折叠，两侧为α螺旋。 的 活性位点，位于β折叠的C-末端边缘， 在细菌家族中高度保守的残基 响应调节器。 活性位点酸性口袋，由一簇 羧酸酯侧链，是磷酸化和二价金属的位点 离子结合 使用CheY作为模型调节域， 磷酸转移和去磷酸化将通过构建 位点特异性突变，表征 这些改变的蛋白质的磷酸化/去磷酸化活性 使用小分子磷酸供体，并确定 通过X射线晶体学分析相关突变蛋白。 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 磷酸供体能够磷酸化晶体中的CheY。 的方向迈出了一步 以确定调节效应器功能的机制， 开关结构域的磷酸化，两个额外的 与甲基酯酶（切布）和DNA结合的反应调节剂 （OmpR）效应子结构域将被启动。

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.