Synthesis of phosphono-CheY and phosphono-VHR

膦酰基-CheY 和膦酰基-VHR 的合成

• 批准号: 7882055
• 负责人: CHRISTOPHER John HALKIDES
• 金额: $ 21.6万
• 依托单位: UNIVERSITY OF NORTH CAROLINA WILMINGTON
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-03-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7882055
• 关键词: Acids; Active Sites; Bacteria; Binding; Chemicals; Chemotaxis; Complex; Crystallography; Cysteine; Data; Diabetes Mellitus; Drug Design; Environment; Enzymes; Event; Family; Flagella; Goals; Health; Human; Hydrolysis; Ions; Lead; Left; Longevity; Malignant Neoplasms; Mammals; Mediating; Metals; Modification; Molecular Conformation; Motor; Organism; Pathogenesis; Pathogenicity; Phosphoproteins; Phosphoric Monoester Hydrolases; Play; Post-Translational Protein Processing; Process; Protein Dephosphorylation; Protein Tyrosine Phosphatase; Proteins; Reaction; Reagent; Research; Role; Signal Transduction; Signal Transduction Pathway; Sodium Chloride; Specificity; Structure; Students; Surface; Swimming; System; Vaccinia; Water; Work; analog; base; cell growth; cell motility; computerized data processing; design; human disease; inhibitor/antagonist; inorganic phosphate; insight; interest; liquid chromatography mass spectrometry; mutant; novel; programs; protein structure; public health relevance; response

DESCRIPTION (provided by applicant): The goal of this project is to determine the structures of two signal transduction proteins, Che-Y and VHR, that are potentially relevant to human diseases. The unique strategy of this work is the use of chemical modification of the proteins to create stable analogs of transient, phosphorylated forms of each protein that would be otherwise difficult to study. Specifically, a cysteine residue is modified with a phosphonomethyl group, CH2PO3. The presence of the phosphonomethylcysteine residue will be confirmed by liquid chromatography and mass spectrometry. The structures of the proteins will be determined by x-ray crystallography. The first protein is CheY, a protein necessary for bacteria to swim toward a more desirable environment, a process known as chemotaxis. Phosphono-CheY was synthesized from a mutant form of CheY and phosphonomethytrifluoromethanesulfonate. The structure of phosphono-CheY will be determined in complex with its partner, FliM. The structure of phosphono-CheY complexed with phosphatases designed to return CheY to its dephosphorylated state may also be determined to obtain insight into the signal termination process. Disruption of chemotaxis may render an organism less pathogenic. The second protein is VHR, a dual-specificity phosphatase. Dysregulation of protein tyrosine phosphatases plays a role in many human diseases, including cancer and diabetes. Protein tyrosine phosphatase enzymes break down phosphoproteins via the creation of a cysteinyl phosphate intermediate that is subsequently hydrolyzed. VHR is inactivated when the cysteine residue is modified with a phosphonomethyl group to become phosphonomethylcysteine. Reversible inhibitors protect against inactivation. Determining the structure of a stable analog of this phosphonomethylcysteine intermediate may provide information about the mechanism of this reaction and may provide a target for structure-based drug design. PUBLIC HEALTH RELEVANCE: This project may lead to a better understanding of how to decrease bacterial pathogenesis by interfering with bacterial motility. Further, this project will provide insights into how cellular growth signals are turned on and off. Errors in these signaling processes underlie many human diseases, including diabetes.

描述（由申请人提供）：该项目的目标是确定与人类疾病潜在相关的两种信号转导蛋白Che-Y和VHR的结构。这项工作的独特策略是利用蛋白质的化学修饰来创建稳定的瞬时磷酸化形式的蛋白质类似物，否则很难研究。具体地说，半胱氨酸残基被磷甲乙基CH2PO3修饰。磷酸亚甲基半胱氨酸残基的存在将通过液相色谱和质谱法证实。蛋白质的结构将由x射线晶体学测定。第一种蛋白质是CheY，一种细菌向更理想的环境游动所必需的蛋白质，这一过程被称为趋化性。由CheY和磷酸甲基三氟甲烷磺酸盐的突变体合成了Phosphono-CheY。膦- chey的结构将在配合物FliM中确定。phosphono-CheY与旨在使CheY返回其去磷酸化状态的磷酸酶络合的结构也可以被确定，以深入了解信号终止过程。趋化性的破坏可以降低生物体的致病性。第二种蛋白质是VHR，一种双特异性磷酸酶。蛋白酪氨酸磷酸酶的失调在许多人类疾病中起作用，包括癌症和糖尿病。蛋白质酪氨酸磷酸酶通过产生半胱氨酸磷酸盐中间体来分解磷酸化蛋白，该中间体随后被水解。当半胱氨酸残基被磷甲酰基修饰成磷甲酰基半胱氨酸时，VHR失活。可逆抑制剂防止失活。确定这种膦甲基半胱氨酸中间体的稳定类似物的结构可能提供有关该反应机制的信息，并可能为基于结构的药物设计提供靶标。

项目成果

The structures of T87I phosphono-CheY and T87I/Y106W phosphono-CheY help to explain their binding affinities to the FliM and CheZ peptides.

• DOI: 10.1016/j.abb.2008.08.019
• 发表时间: 2008-11-15
• 期刊: Archives of biochemistry and biophysics
• 影响因子: 3.9
• 作者: McAdams K;Casper ES;Matthew Haas R;Santarsiero BD;Eggler AL;Mesecar A;Halkides CJ
• 通讯作者: Halkides CJ