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Structure-function study of blood coagulation factor VII by in vitro mutagenesis and computer simulation.

机译：通过体外诱变和计算机模拟研究凝血因子VII的结构功能。

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摘要

Coagulation factor VIIa (FVIIa) is a plasma trypsin-like protease. It consists of a γ-carboxyl glutamic acid domain (Gla), 2 epidermal growth factor-like domains (Egf1 and Egf2) and a catalytic domain at its C-terminus. Activation of FVII to FVIIa, followed by binding to its cofactor, tissue factor (TF), is the first step in the extrinsic pathway of the blood coagulation cascade. I investigated the following questions: what FVII residues are important for activation, what FVIIa residues are involved in macromolecular substrate binding and what is the mechanism by which TF enhances FVIIa's activity? To address these questions, I used molecular simulation and FVIIa 3-D structure information to identify potentially important residues and employed molecular biological methods to create mutant FVII's. I found a hydrophobic patch on the surface of FVII's catalytic domain which is specifically involved in the activation of FVII by factor Xa (FXa). Mutation of two residues in this patch, H101 and Y179 to alanine (H101A, Y179A) decreased the catalytic efficiencies of FXa in FVII activation by 15- and 8-fold respectively. Activation of FVII with multiple mutations including both H101A and Y179A by FXa was not measurable. Furthermore, I identified an exosite involved in FX and FIX binding on the surface of FVIIa, about 90° from the hydrophobic patch. Mutations at this exosite, L144Aa and R147Aa, had kcat's lower than wild-type by 40- and 4-fold respectively toward FX Activation of FIX was also affected by these mutations, although less significantly than FX activation. A FVIIa with mutations including both L144A and R147 had almost no activity for FX or FIX activation, indicating that FX and FIX share at least part of the same binding site on FVIIa's surface. Because the Egf1 domain contributes to TF binding, I used FVII with its Egf1 domain substituted with FIX's (FVII IXegf1) to study Egf1's function. in addition to TF binding, the catalytic activity of FVIIa was affected. Mutation of the residue (lysine) at position 79 back to FVII's (arginine) regained some functions both in TF binding affinity and its catalytic activity. The affected catalytic activities were displayed only in the presence of TF. My hypothesis is that the Egf1 substitution effect is transmitted through the interface between the catalytic domain and the N-terminus of TF. To test this hypothesis, I carried out dynamics simulation of FVIIa-TF and FVIIIXegf1a-TF complexes. The results showed that 3 loops, with residues 310–329, residues 333–337 and residues 369–374, were deformed and shifted in position due to the disruption of the catalytic domain-TF interface. This affected both FVII's substrate binding sites and the hydrophobic environment required for stabilizing the N-terminus insertion of the catalytic domain of FVIIa. The simulations suggest that any changes in the local conformation of these loops or their neighborhood may affect catalytic activity. This prediction is supported by the evidence from in vitro mutagenesis.
机译：凝血因子VIIa（FVIIa）是血浆胰蛋白酶样蛋白酶。它由一个γ-羧基谷氨酸结构域（Gla），两个表皮生长因子样结构域（Egf1和Egf2）和一个位于其C末端的催化结构域组成。 FVII活化为FVIIa，然后结合其辅因子组织因子（TF），是凝血级联外部途径中的第一步。我研究了以下问题：哪些FVII残基对于激活很重要，哪些FVIIa残基参与大分子底物结合，TF增强FVIIa活性的机制是什么？为了解决这些问题，我使用了分子模拟和FVIIa 3-D结构信息来识别潜在的重要残基，并采用了分子生物学方法来创建突变FVII。我在FVII催化结构域的表面上发现了一个疏水补丁，该补丁专门参与因子Xa（FXa）激活FVII。该补丁中的两个残基H101和Y179突变为丙氨酸（H101A，Y179A）使FXa在FVII激活中的催化效率分别降低了15倍和8倍。无法测量FXa对具有H101A和Y179A多个突变的FVII的激活。此外，我鉴定了一个与FVIIa表面上的FX和FIX结合的异位点，该位点与疏水性贴剂相距约90°。 L144Aa和R147Aa这个外位点的突变使 k cat 分别比野生型低40倍和4倍，对FX的激活FIX的激活也受到影响这些突变，尽管不如FX激活显着。具有突变的FVIIa（包括L144A和R147）几乎没有激活FX或FIX的活性，这表明FX和FIX至少共享FVIIa表面上相同结合位点的至少一部分。由于Egf1结构域有助于TF结合，因此我使用FVII，将其Egf1结构域替换为FIX（FVII IXegf1 ），以研究Egf1的功能。除TF结合外，FVIIa的催化活性也受到影响。 79位残基（赖氨酸）突变回到FVII（精氨酸），重新获得了TF结合亲和力和催化活性方面的某些功能。仅在TF存在下显示受影响的催化活性。我的假设是Egf1取代作用是通过催化域和TF N端之间的界面传递的。为了验证该假设，我对FVIIa-TF和FVII IXegf1 a-TF复合物进行了动力学模拟。结果表明，由于催化域-TF界面的破坏，残基为310-329，残基333-337和残基369-374的3个环发生了变形并移位。这影响了FVII的底物结合位点和稳定FVIIa催化结构域的N端插入所需的疏水环境。模拟表明这些环或其附近的局部构象的任何变化都可能影响催化活性。体外诱变的证据支持了这一预测。

著录项

  • 作者

    Jin, Jianping.;

  • 作者单位

    The University of North Carolina at Chapel Hill.;

  • 授予单位 The University of North Carolina at Chapel Hill.;
  • 学科 Biology Molecular.; Chemistry Biochemistry.; Computer Science.
  • 学位 Ph.D.
  • 年度 1999
  • 页码 114 p.
  • 总页数 114
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 分子遗传学;生物化学;自动化技术、计算机技术;
  • 关键词

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