Structural Basis of Serpin Function and Regulation

Serpin 功能和调节的结构基础

• 批准号: 7329181
• 负责人: Steven T. Olson
• 金额: $ 36.74万
• 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/7329181
• 关键词: Affect; Antithrombins; Apoptosis; Binding; Binding Proteins; Blood coagulation; Burial; Chimera organism; Class; Collaborations; Complex; Coupling; Crystallization; Cysteine Protease; Dependence; Disruption; Disulfides; Docking; Elements; Endopeptidases; Engineering; Equilibrium; Factor Xa; Family; Fibrinolysis; Fluorescence; Fluorescence Resonance Energy Transfer; Goals; Helix (Snails); Inflammation; Kinetics; Knowledge; Label; Lead; Ligands; Mediating; Membrane; Molecular Conformation; Movement; Mutagenesis; Mutate; Mutation; N-terminal; Object Attachment; 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; ear helix; 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.

说明书(申请人提供)：丝氨酸蛋白超家族的蛋白水解酶抑制剂在调节细胞内外丝氨酸和半胱氨酸蛋白酶的许多生理过程中起着重要的作用。Serpins通过一种新的抑制机制来调节蛋白水解酶，在该机制中，由于丝氨酸和蛋白水解酶的主要构象变化，该酶被捕获在Serpin蛋白分解的酰基中间阶段作为常规底物。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一机制的悬而未决的问题。从这些研究中获得的知识有望加强对蛇调控蛋白分解的复杂模式的理解，并为天然蛇突变如何破坏这一调控提供新的见解。这些研究将验证以下假设：1)蛇毒通过不同的机制捕获稳定的酰基中间体复合体中不同结构家族的蛋白酶；2)丝氨酸F螺旋在丝氨酸抑制机制中发挥重要的活性作用；3)Z依赖的丝氨酸ZPI通过与蛋白Z结合并通过位于蛋白Z和ZPI中的外位相互作用识别膜结合因子Xa来调节促凝血剂复合体中的Xa因子。这些假说将通过以下具体目标得到验证：1)我们将评估I)破坏丝氨酸反应环与蛋白酶的相互作用，II)扭曲蛋白酶，以及III)扭曲的蛋白酶与丝氨酸结合在稳定不同机械类别、结构和特异性的蛋白酶的丝氨酸-蛋白酶酰基中间复合体中的相对重要性；2)我们将阐明F螺旋是否在通过可逆的运动或螺旋结构变化将丝氨酸反应环的构象变化的能量耦合到扭曲酰基-中间复合体的能量中发挥积极作用；3)我们将确定调节Xa因子特异的丝蛋白ZPI中丝蛋白特异性和丝蛋白功能调节的结构要求。建议的研究将利用突变、荧光、核磁共振和X射线结晶学以及热力学和动力学方法来表征丝氨酸蛋白酶和丝氨酸蛋白酶与配体的相互作用。