STRUCTURE/FUNCTION ANALYSIS OF MITOTIC REGULATORS

有丝分裂调节因子的结构/功能分析

• 批准号: 6180560
• 负责人: Joseph Patrick Noel
• 金额: $ 26.87万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-05-01 至 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6180560
• 关键词: HeLa cells; X ray crystallography; active sites; cell cycle; cell growth regulation; cyclin dependent kinase; enzyme inhibitors; enzyme mechanism; enzyme structure; enzyme substrate; fluorescence spectrometry; isozymes; peptidylprolyl isomerase; stereochemistry; surface plasmon resonance

Our major goal is to provide a stereochemical foundation for the biological roles played by a novel mitotic regulator known as Pin1. The human peptidyl-prolyl cis-trans isomerase (PPIase) Pin1 is a conserved mitotic regulator essential for the G2 to M transition of the eukaryotic cell cycle. We have determined the high resolution x-ray crystal structure of Pin1 complexed with a proline containing substrate. In addition, we have begun a functional analysis of Pin1 in vitro and in vivo in order to correlate the observed structural features of this novel PPIase with its role in regulating entry and exit from mitosis. The crystallographic structure together with pH titration studies and mutagenesis of an active site cysteine suggest a catalytic mechanism that includes general acid-base and covalent catalysis during peptide bond isomerization. Pin1 displays a strong preference for an acidic residue N-terminal to the isomerized proline bond due to interaction of this acidic side chain with a conserved basic cluster at the entrance to the PPIase domain's active site. Structural work in progress, confirms that optimal interaction with this basic cluster is achieved with phosphothreonine or phosphoserine containing substrates. The known specificity of cyclin-dependent protein kinases (CDKs) is for threonine or serine residues amino terminal to proline. Pin1, with an active site structure ideally suited for binding phosphorylated -SP- or -TP- containing substrates, likely regulates CDK targets during progression from the inter- to the mitotic phase of the cell cycle. In particular, PinI directly regulates the activity of the mitotic phosphatase CDC25C by binding to its hyperphosphorylated N-terminal region. This region rich in CDK sites, is phosphorylated, and this phosphorylation is essential for CDC25C activity. With regard to this proposal, we will employ a two tiered approach involving a combination of macromolecular x-ray crystallography for high resolution structural analysis of Pin1 and a number of Pin1 mutants in complex with substrates including CDC25C, and in vitro functional analysis of Pin1's substrate specificity and mechanism of action. In addition to the structural work, we will determine the kinetic and thermodynamic parameters governing Pin1-target protein interactions. In addition, we will complete the structural elucidation of the tetramerization domain of a Pin1 interacting kinase known as NIMA and begin crystallization and structural elucidation of Pin1 complexed to this domain of the NIMA kinase. Finally, we propose to design, synthesize and test several structurally based Pin1 inhibitors as new anti-mitotic agents.

我们的主要目标是提供一个立体化学的基础， 一种新的有丝分裂调节因子Pin 1所起的生物学作用。 的 人肽基脯氨酰顺反异构酶（PPIase）Pin 1是一种保守的 对真核细胞的G2到M转变至关重要的有丝分裂调节因子 细胞周期 我们已经确定了高分辨率的x射线晶体 与含脯氨酸的底物复合的Pin 1的结构。 在 此外，我们已经开始在体外和体内对Pin 1进行功能分析， 为了将观察到的这种结构特征与 具有调节有丝分裂进入和退出的作用的新型PPIase。 晶体结构连同pH滴定研究， 活性位点半胱氨酸诱变提示催化机制 包括一般的酸碱和共价催化作用， 键异构化 Pin 1显示出对酸性的强烈偏好。 由于以下物质的相互作用，异构化脯氨酸键的N-末端残基 这个酸性侧链在入口处有一个保守的碱性簇， PPIase结构域的活性位点 正在进行结构性工作， 证实实现了与该基本群集的最佳交互 与含磷酸苏氨酸或磷酸丝氨酸的底物反应。 已知 细胞周期蛋白依赖性蛋白激酶（CDKs）的特异性是针对苏氨酸 或脯氨酸氨基末端的丝氨酸残基。Pin 1，带有活动站点 理想地适合于结合磷酸化的-SP-或-TP-的结构 含有底物，可能在进展过程中调节CDK靶点 从细胞周期的间期到有丝分裂期。 特别是， PinI直接调节有丝分裂磷酸酶CDC 25 C的活性 通过结合其高度磷酸化的N-末端区域。 这一地区 富含CDK位点，是磷酸化的，这种磷酸化是 对CDC 25 C活性至关重要。 关于这项建议，我们会 采用两层方法，包括大分子 Pin 1高分辨结构的X射线晶体学分析 和许多与底物复合的Pin 1突变体，包括 CDC 25 C，以及Pin 1底物特异性的体外功能分析 和作用机制。 除了结构性工作，我们还将 确定控制Pin 1-target的动力学和热力学参数 蛋白质相互作用 此外，我们将完成结构 Pin 1相互作用激酶的四聚化结构域的阐明 并开始结晶和结构解析 Pin 1与NIMA激酶的该结构域复合。 最后提出 设计、合成和测试了几种结构上基于Pin 1 抑制剂作为新的抗有丝分裂剂。