The second Kunitz domain of human tissue factor pathway inhibitor: Cloning, structure determination and interaction with Factor Xa
The second Kunitz domain of human tissue factor pathway inhibitor: Cloning, structure determination and interaction with Factor Xa
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DOI：
10.1006/jmbi.1997.1029
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发表时间：
1997-06-13
影响因子：
5.6
通讯作者：
Stubbs, MT
中科院分区：
文献类型：
--
作者：
Burgering, MJM;Orbons, LPM;Stubbs, MT
组织因子途径抑制剂（TFPI）是一种36kda的糖蛋白，通过抑制Xa因子和VIIa因子/组织因子（TF）复合物来帮助维持止血。TFPI包含三个串联的Kunitz抑制剂结构域，其中第二个抑制因子Xa。我们采取了多学科的方法来研究TFPI的第二个Kunitz结构域的结构和功能，以期合理设计Xa因子抑制剂。成熟TFPI蛋白第2 Kunitz结构域（TFPI- kii）的第93 ~ 154个氨基酸残基在大肠杆菌中表达。通过离子交换、疏水相互作用和尺寸排除层析纯化得到接近均匀的蛋白。TFPI- kii是一种有效的Xa因子抑制剂，其K-i值为1.5 x 10(-10) M，与完整的TFPI没有显著差异。采用H-1核磁共振波谱法（NMR）测定了TFPI-kII在水溶液中的三维结构。用DIANA程序计算了一组30个构象，使用了906个由核Overhauser效应导出的距离约束和23个二面角约束。该集合代表了TFPI-kII的溶液结构，主原子的平均均方根偏差为0.78埃，残数1 ~ 58的所有重原子的平均均方根偏差为1.38埃。利用x射线晶体学技术也确定了TFPI-kII与猪胰蛋白酶配合物的结构。该配合物的解算分辨率为2.6埃，最终r因子为16.2%。与胰蛋白酶配合物中TFPI-kII的核磁共振衍生结构比较发现，除了残基Tyr17外，两者结构差异不大。胰蛋白酶：TFPI-kII复合物在Xa因子上的叠加提供了对Xa因子抑制的大分子决定因素的见解。络合需要一定程度的因子Xa残基重组，尤其是TyrF99，但也可能需要f148环的重组。用约束分子动力学进一步研究了这种相互作用。静电相互作用似乎起了主要作用。因子Xa的重组与提出的因子Xa:TAP相互作用形成对比，其中TAP将与因子Xa的“基态”结构结合。(C) 1997学术出版社有限公司
Tissue Factor Pathway Inhibitor (TFPI) is a 36 kDa glycoprotein that helps maintain haemostasis by inhibiting Factor Xa and the Factor VIIa/Tissue Factor (TF) complex. TFPI contains three tandemly Linked Kunitz inhibitor domains, of which the second inhibits factor Xa. We have undertaken a multidisciplinary approach to study the structure and function of the second Kunitz domain of TFPI, with a view towards the rational design of factor Xa inhibitors. Amino acid residues 93 to 154 of the mature TFPI protein, corresponding to the second Kunitz domain (TFPI-kII), were expressed in Escherichia coli. The protein was purified to near homogeneity by ion exchange, hydrophobic interaction, and size exclusion chromatography, respectively. TFPI-kII is a potent factor Xa inhibitor with a K-i of 1.5 x 10(-10) M, a value that does not differ significantly from that of intact TFPI. The three-dimensional structure of TFPI-kII in aqueous solution was determined by H-1 nuclear magnetic resonance spectroscopy (NMR). A set of 30 conformers was calculated with the program DIANA using 906 distance constraints derived from nuclear Overhauser effects and 23 dihedral angle constraints. This set, representing the solution structure of TFPI-kII, has an average root-mean-square deviation of 0.78 Angstrom for the backbone atoms and 1.38 Angstrom for all heavy atoms of residues 1 to 58. The structure of TFPI-kII has also been determined in complex with porcine trypsin using X-ray crystallographic techniques. The complex has been solved to a resolution of 2.6 Angstrom, with a final R-factor of 16.2%. Comparison of the NMR derived structure with that of TFPI-kII in complex with trypsin reveals Little divergence of the two structures, with the exception of residue Tyr17. Superposition of the trypsin:TFPI-kII complex on factor Xa provides insights into macromolecular determinants for the inhibition of factor Xa. Complexation would require a degree of reorganisation of factor Xa residues, in particular of TyrF99, but also perhaps of the F148-loop. The interaction was further investigated using restrained molecular dynamics. Electrostatic interactions would appear to play a major role. The reorganisation of factor Xa is in contrast to the proposed factor Xa:TAP interaction, where TAP would bind to the ''ground state'' structure of factor Xa. (C) 1997 Academic Press Limited.