Structure-function analysis of the reactive site in the first Kunitz-type domain of human tissue factor pathway inhibitor-2
Structure-function analysis of the reactive site in the first Kunitz-type domain of human tissue factor pathway inhibitor-2
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DOI：
10.1074/jbc.m400802200
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发表时间：
2004-04-23
影响因子：
4.8
通讯作者：
Kisiel, W
中科院分区：
文献类型：
--
作者：
Chand, HS;Schmidt, AE;Kisiel, W
人组织因子途径抑制因子-2(TFPI-2)是一种库尼茨类型的蛋白酶抑制物，通过与第一个库尼茨类型结构域(Kd1)上的P-1残基(Arg-24)的非生产性相互作用，调节参与凝血和纤溶的多种丝氨酸蛋白酶。以前的动力学研究表明，TFPI-2是一种比其他几种丝氨酸蛋白酶更有效的纤溶酶抑制物，但这种特异性的分子基础尚不清楚。在本研究中，我们使用分子建模和突变策略来产生人TFPI-2Kd1的几个变体，以努力确定P-1Arg以外的相互作用位点残基对其抑制活性和特异性有显著影响。基于牛胰酶抑制剂晶体结构对Kd1进行的分子模拟表明，Kd1与纤溶酶和胰酶形成能量更有利的复合体和/或因子VIIa-组织因子复合体，主要是由于Asp-19(P-6)与纤溶酶中Arg残基(Arg-644、Arg-719和Arg-767)、Arg-24(P-1)与Asp-735以及Arg-29(P-5‘)与纤溶酶中Glu-606残基之间的强离子相互作用。此外，Kd1中的Leu-26至Leu-28(P-2‘-P-4’)与纤溶酶中的疏水簇(Phe-583、Met-585和Phe-587)形成了较强的van der Waals接触。与野生型Kd1相比，Asp-19、Tyr-20、Arg-24、Arg-29和Leu-26的突变导致纤溶酶抑制活性显著降低，但Asp-19和Tyr-20突变揭示了这些残基在纤溶酶特异性抑制中的重要性。除了Kd1的P-6-P-5‘区的反应位点残基外，P-18’位高度保守的Phe的突变揭示了该残基在Kd1抑制丝氨酸蛋白酶中的重要性。因此，与P-1残基一起，P-1残基两侧的其他残基的性质，特别是P-6和P-5‘处的残基的性质，强烈影响人TFPI-2的抑制活性和特异性。
Human tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type proteinase inhibitor that regulates a variety of serine proteinases involved in coagulation and fibrinolysis through their non-productive interaction with a P-1 residue (Arg-24) in its first Kunitz-type domain (KD1). Previous kinetic studies revealed that TFPI-2 was a more effective inhibitor of plasmin than several other serine proteinases, but the molecular basis for this specificity was unclear. In this study, we employed molecular modeling and mutagenesis strategies to produce several variants of human TFPI-2 KD1 in an effort to identify interactive site residues other than the P-1 Arg that contribute significantly to its inhibitory activity and specificity. Molecular modeling of KD1 based on the crystal structure of bovine pancreatic trypsin inhibitor revealed that KD1 formed a more energetically favorable complex with plasmin versus trypsin and/or the factor VIIa-tissue factor complex primarily due to strong ionic interactions between Asp-19 (P-6) and Arg residues in plasmin (Arg-644, Arg-719, and Arg-767), Arg-24 (P-1) with Asp- 735 in plasmin, and Arg-29 (P-5') with Glu-606 in plasmin. In addition, Leu-26 through Leu-28 (P-2'-P-4') in KD1 formed strong van der Waals contact with a hydrophobic cluster in plasmin (Phe-583, Met-585, and Phe-587). Mutagenesis of Asp-19, Tyr-20, Arg-24, Arg-29, and Leu-26 in KD1 resulted in substantial reductions in plasmin inhibitory activity relative to wild-type KD1, but the Asp-19 and Tyr-20 mutations revealed the importance of these residues in the specific inhibition of plasmin. In addition to the reactive site residues in the P-6-P-5' region of KD1, mutation of a highly conserved Phe at the P-18' position revealed the importance of this residue in the inhibition of serine proteinases by KD1. Thus, together with the P-1 residue, the nature of other residues flanking the P-1 residue, particularly at P-6 and P-5', strongly influences the inhibitory activity and specificity of human TFPI-2.