STRUCTURE/FUNCTION ANALYSIS OF MITOTIC REGULATORS

我们的主要目标是为 一种名为Pin1的新的有丝分裂调节因子所发挥的生物学作用。这个 人肽基-脯氨酰顺-反式异构酶(PPIase)Pin1是一个保守的 真核细胞G2向M期转变所必需的有丝分裂调控因子 细胞周期。我们已经确定了高分辨率的x射线晶体 Pin1与含有底物的脯氨酸络合的结构。在……里面 此外，我们已经开始在体外和体内对Pin1进行功能分析。 为了将观察到的这一结构特征 新的PPIase及其在调节有丝分裂进出中的作用。 晶体结构与pH滴定研究和 半胱氨酸活性部位的突变提示催化机制 这包括多肽过程中的一般酸碱催化和共价催化 键异构化。Pin1表现出强烈的酸性偏好 与异构化的Pro键相互作用所致的残基N-末端 这条酸性侧链的入口处有一个保守的碱性簇 PPIase域的活动站点。正在进行的结构工作， 确认已实现与此基本集群的最佳交互 含有磷苏氨酸或磷丝氨酸的底物。已知的 细胞周期蛋白依赖性蛋白激酶(CDKs)对苏氨酸的特异性 或丝氨酸残基的氨基末端到脯氨酸。PIN1，具有活动站点 结构非常适合于结合磷酸化的SP或TP- 含有底物，可能在进展过程中调节CDK靶点 从细胞周期的分裂间期到有丝分裂期。特别是， PINI直接调节有丝分裂磷酸酶CDC25C的活性 通过与其过度磷酸化的N-末端区域结合。这一地区 富含CDK位点，是磷酸化的，这种磷酸化是 对CDC25C活性至关重要。关于这项建议，我们将 采用两个层次的方法，包括组合大分子 X射线结晶学用于PIN1的高分辨结构分析 和一些Pin1突变体与底物的复合体，包括 CDC25C，以及Pin1‘S底物特异性的体外功能分析 和作用机制。除了结构工作外，我们还将 确定控制Pin1靶的动力学和热力学参数 蛋白质的相互作用。此外，我们还将完成结构 Pin1相互作用蛋白激酶四聚化结构域的研究 被称为尼玛，并开始结晶和结构阐明 Pin1与NIMA激酶的这个结构域形成复合体。最后，我们建议 设计、合成和测试几种基于结构的PIN1 作为新的抗有丝分裂药物的抑制剂。

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.