STRUCTURE AND FUNCTION OF RESPONSE REGULATOR PROTEINS

反应调节蛋白的结构和功能

• 批准号: 2185379
• 负责人: ANN M. STOCK
• 金额: $ 18.31万
• 依托单位: UNIV OF MED/DENT NJ-R W JOHNSON MED SCH
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-01 至 1997-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2185379
• 关键词: DNA binding protein; Escherichia coli; X ray crystallography; bacterial genetics; biological signal transduction; chemotaxis; conformation; esterase; fluorescence; gene mutation; mutant; phosphorylation; phosphotransferases; polymerase chain reaction; protein structure function; site directed mutagenesis; spectrometry

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.

在真核生物和原核的各种反应的调节 细胞涉及蛋白质的家族，这些蛋白质可作为分子开关的蛋白质 打开和关闭特定效应子功能。 不活动之间的切换 主动状态是由构象变化引起的 通过蛋白质侧链的共价修改或结合 小分子。 在核细胞中，一个响应调节剂家族 由同源开关域和特定效应子组成的蛋白质 域，调解对改变环境条件的反应。 这 法规域通过天冬氨酸侧的磷酸化打开 链条并通过固有的酰基磷酸盐水解关闭 磷酸酶活性。 调节结构域的磷酸化是通过 来自相关组氨酸蛋白激酶的磷酸转移。 这些 磷酸转移介导的信号转导系统是广泛的 在整个细菌王国和调节过程（例如细胞） 运动，差异化，运输，代谢和建立 宿主/病原体相互作用。 拟议的研究重点 这些磷酸化激活开关的结构/功能分析 域的目的是了解其分子机制 行动。 具体而言，磷酸转移和 磷酸盐水解？ 构象变化的本质是什么 是由磷酸化诱导的吗？ 调节域如何传输 它对效应域的影响？ 细菌趋化蛋白， 调节鞭毛旋转的Chey是代表 磷酸化激活的调节域。 这个128个氨基酸单 域蛋白具有经典的α/beta折叠，由中央五个 滞留的平行β板在两侧的alpha螺旋侧两侧。 这 位于Beta表的C末端边缘的活动位置组成 在细菌家族中高度保守的残留物 响应调节器。 由一个活跃的位点酸性口袋，由一群 羧酸盐侧链是磷酸化和二价金属的位置 离子结合。 使用Chey作为模型调节域， 磷酸化和去磷酸化将通过构造来解决 特定地点突变，表征 这些改变的蛋白质的磷酸化/去磷酸化活性 使用小分子磷酸化剂，并确定 相关突变蛋白通过X射线晶体学。 X射线结构 将通过筛选来接近Chey的主动构象 稳定活性形式的突变，并使用小分子 能够在晶体中磷酸化的Chey的磷酸化。 作为一步 通过确定通过 开关域的磷酸化，两个附加的结构分析 具有附着甲基酯酶（CHEB）和DNA结合的响应调节剂 （OMPR）将启动效应域。