Structural Basis of Serpin Function and Regulation

描述（由申请人提供）：丝氨酸蛋白超家族的蛋白蛋白酶抑制剂在许多生理过程中调节细胞内和细胞外丝氨酸和半胱氨酸蛋白酶发挥重要作用。蛇形蛋白通过一种新的抑制机制来调节蛋白水解酶，在这种机制中，由于蛇形蛋白和蛋白酶的主要构象变化，蛋白酶被困在蛇形蛋白水解的酰基-中间阶段，作为一种规则的底物。提出的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悬而未决的问题。从这些研究中获得的知识有望增强对蛇蛋白调节蛋白质水解的复杂模式的理解，并为自然蛇蛋白突变如何破坏这种调节提供新的见解。拟开展的研究将验证以下假设：1)蛇肽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-中间复合物中；ii)蛇形蛋白F螺旋在蛇形蛋白抑制机制中起着重要的积极作用；iii)蛋白Z依赖性蛇形蛋白ZPI通过结合蛋白Z并通过蛋白Z和ZPI的外源相互作用识别膜结合因子Xa来调节促凝物复合体中的Xa因子。这些假设将通过以下具体目标进行测试：1)我们将评估以下方面的相对重要性：1)破坏丝氨酸蛋白酶反应性环-蛋白酶相互作用；2)扭曲蛋白酶；3)扭曲蛋白酶与丝氨酸的结合在稳定不同机制类别、结构和特异性的丝氨酸蛋白酶酰基中间复合物中的作用；2)我们将阐明F螺旋是否在丝氨酸反应环构象变化的能量耦合中发挥积极作用，从而通过螺旋的可逆运动或结构变化扭曲酰基-中间体中的蛋白酶；3)我们将确定介导serpin特异性的结构要求以及因子xa特异性serpin ZPI中serpin功能的调节。该研究将利用诱变、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蛇形蛋白蛋白酶和蛇形蛋白配体的相互作用。

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.