Structural Basis of Serpin Function and Regulation

Serpin 功能和调节的结构基础

• 批准号: 6999372
• 负责人: Steven T. Olson
• 金额: $ 37.84万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-12-27 至 2009-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6999372
• 关键词: X ray crystallography; active sites; conformation; enzyme induction /repression; enzyme mechanism; enzyme structure; ligands; nuclear magnetic resonance spectroscopy; protease inhibitor; protein binding; protein protein interaction; protein structure function; proteolysis; serine proteinases; structural biology

DESCRIPTION (provided by applicant): Protein protease inhibitors of the serpin superfamily play important roles in regulating intracellular and extracellular serine and cysteine proteases in numerous physiologic processes. Serpins regulate proteolytic enzymes through a novel inhibition mechanism in which the protease is trapped at the acyl-intermediate stage of proteolysis of the serpin as a regular substrate due to major conformational changes induced in both the serpin and protease. The long term goal of the proposed studies is to answer outstanding questions concerning this unusual conformational trapping mechanism and how various accessory ligands modulate this mechanism. The knowledge gained from these studies is expected to enhance understanding of the complex modes by which serpins regulate proteolysis and provide new insights into how natural serpin mutations disrupt this regulation. The proposed studies will test the hypotheses that i) serpins trap proteases of different structural families in stable acyl-intermediate complexes by different mechanisms; ii) the serpin F helix plays an essential active role in the serpin inhibitory mechanism and iii) the protein Z-dependent serpin, ZPI, regulates factor Xa in procoagulant complexes by binding protein Z and recognizing membrane-bound factor Xa through exosite interactions residing in protein Z and ZPI. These hypotheses will be tested by the following specific aims: 1) we will assess the relative importance of i) disrupting serpin reactive loop-protease interactions, ii) distorting the protease and iii) binding of the distorted protease to the serpin in stabilizing serpin-protease acyl-intermediate complexes for proteases of different mechanistic class, structure and specificity; 2) we will elucidate whether the F helix plays an active role in coupling the energy of the serpin reactive loop conformational change to distorting the protease in the acyl-intermediate complex through reversible movements or structural changes in the helix; and 3) we will determine the structural requirements which mediate serpin specificity and the regulation of serpin function in the factor Xa-specific serpin, ZPI. The proposed studies will utilize mutagenesis, fluorescence, NMR and X-ray crystallography and thermodynamic and kinetic approaches to characterize serpin-protease and serpin-ligand interactions.

描述（由申请人提供）：丝氨酸蛋白酶抑制剂超家族的蛋白质蛋白酶抑制剂在调节细胞内和细胞外丝氨酸和半胱氨酸蛋白酶在许多生理过程中发挥重要作用。丝氨酸蛋白酶抑制剂通过一种新的抑制机制调节蛋白水解酶，其中由于丝氨酸蛋白酶抑制剂和蛋白酶中诱导的主要构象变化，蛋白酶作为常规底物被捕获在丝氨酸蛋白酶抑制剂的蛋白水解的酰基中间阶段。拟议研究的长期目标是回答有关这种不寻常的构象捕获机制以及各种辅助配体如何调节这种机制的突出问题。从这些研究中获得的知识预计将增强对丝氨酸蛋白酶抑制剂调节蛋白水解的复杂模式的理解，并为天然丝氨酸蛋白酶抑制剂突变如何破坏这种调节提供新的见解。拟议的研究将检验以下假设：i) 丝氨酸蛋白酶抑制剂通过不同机制将不同结构家族的蛋白酶捕获在稳定的酰基中间体复合物中； ii) 丝氨酸蛋白酶抑制剂 F 螺旋在丝氨酸蛋白酶抑制剂抑制机制中发挥着重要的积极作用，iii) 蛋白质 Z 依赖性丝氨酸蛋白酶抑制剂 (ZPI) 通过结合蛋白质 Z 并通过位于蛋白质 Z 和 ZPI。这些假设将通过以下具体目标进行检验：1）我们将评估 i）破坏丝氨酸蛋白酶抑制剂反应环-蛋白酶相互作用，ii）扭曲蛋白酶和 iii）扭曲蛋白酶与丝氨酸蛋白酶抑制剂的结合在稳定丝氨酸蛋白酶抑制剂中的相对重要性 -用于不同机制类别、结构和特异性的蛋白酶的蛋白酶酰基中间体复合物； 2）我们将阐明F螺旋是否在通过螺旋的可逆运动或结构变化将丝氨酸蛋白酶抑制剂反应环构象变化的能量耦合到酰基中间体复合物中的蛋白酶扭曲方面发挥积极作用； 3)我们将确定介导丝氨酸蛋白酶抑制剂特异性和因子Xa特异性丝氨酸蛋白酶抑制剂ZPI中丝氨酸蛋白酶抑制剂功能调节的结构要求。拟议的研究将利用诱变、荧光、核磁共振和 X 射线晶体学以及热力学和动力学方法来表征丝氨酸蛋白酶抑制剂-蛋白酶和丝氨酸蛋白酶抑制剂-配体相互作用。