Structural Basis of Serpin Function and Regulation

Serpin 功能和调节的结构基础

• 批准号: 7819189
• 负责人: Steven T. Olson
• 金额: $ 0.72万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-08 至 2009-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7819189
• 关键词: Affect; Antithrombins; Apoptosis; Binding; Binding Proteins; Blood coagulation; Burial; Chimera organism; Collaborations; Complex; Coupling; Crystallization; Cysteine Protease; Dependence; Disulfides; Docking; Elements; Engineering; Equilibrium; Factor Xa; Family; Fibrinolysis; Fluorescence; Fluorescence Resonance Energy Transfer; Goals; Inflammation; Kinetics; Knowledge; Label; Lead; Ligands; Mediating; Membrane; Molecular Conformation; Movement; Mutagenesis; Mutate; Mutation; N-terminal; Ovalbumin; Peptide Hydrolases; Physiological Processes; Plasminogen Activator Inhibitor 1; Play; Predisposition; Protease Inhibitor; Proteins; Proteolysis; Recombinants; Regulation; Relative (related person); Reporter; Research Personnel; Role; Serine; Serpin Superfamily; Serpins; Site; Son; Specificity; Staging; Structure; Testing; Thermodynamics; X-Ray Crystallography; base; cofactor; crosslink; deacylation; extracellular; insight; novel; plasma protein Z; programs; serpin-2

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）：Serpin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起着重要作用。 SERPINS通过一种新型的抑制机制来调节蛋白水解酶，其中蛋白酶被蛋白酶捕获在SERPIN蛋白水解的酰基中间阶段，作为正常底物，这是由于SERPIN和蛋白酶引起的重大构象变化。拟议研究的长期目标是回答有关这种异常构象诱捕机制以及各种辅助配体如何调节该机制的杰出问题。从这些研究中获得的知识有望增强对Serpins调节蛋白水解的复杂模式的理解，并提供有关天然SERPIN突变如何破坏这种调节的新见解。拟议的研究将检验假设，即i）通过不同的机制，稳定的酰基中间体复合物中不同结构家族的丝丁陷阱蛋白酶； ii）SERPIN F Helix在SERPIN抑制机制中起着重要的活性作用，iii）蛋白质Z依赖性SERPIN ZPI ZPI，通过结合蛋白质Z并识别膜结合因子Xa通过蛋白质和ZPI中的外来相互作用来调节Procagulant复合物中的Xa。这些假设将通过以下特定目的进行测试：1）我们将评估i）i）破坏塞普蛋白反应性环状 - 蛋白酶相互作用，ii）扭曲蛋白酶和iii）扭曲的蛋白酶与塞普蛋白在稳定serpin-促蛋白酶酰基蛋白酶酰基中间体复合物中的蛋白酶的结合，以使蛋白酶的蛋白酶对不同的机械性类别，结构和特殊性，具体，特殊性，特殊性，特殊性； 2）我们将阐明F螺旋在耦合Serpin反应环构象变化的能量中是否发挥着积极作用，以通过可逆运动或螺旋中的结构变化在酰基中间中间配合物中扭曲蛋白酶； 3）我们将确定介导SERPIN特异性的结构要求以及在Xa特异性SERPIN，ZPI中介导SERPIN功能的调节。拟议的研究将利用诱变，荧光，NMR和X射线晶体学以及热力学和动力学方法来表征Serpin--蛋白酶和Serpin-s-配体相互作用。

项目成果

Kinetic intermediates en route to the final serpin-protease complex: studies of complexes of α1-protease inhibitor with trypsin.

最终丝氨酸蛋白酶抑制剂-蛋白酶复合物途中的动力学中间体：α1-蛋白酶抑制剂与胰蛋白酶复合物的研究。

• DOI: 10.1074/jbc.m113.510990
• 发表时间: 2013
• 期刊: The Journal of biological chemistry
• 作者: Maddur,AshokaA;Swanson,Richard;Izaguirre,Gonzalo;Gettins,PeterGW;Olson,StevenT
• 通讯作者: Olson,StevenT