Factor IXa:factor VIIIA interaction -: Helix 330-338 of factor IX interacts with residues 558-565 and spatially adjacent regions of the A2 subunit of factor VIIIa
Factor IXa:factor VIIIA interaction -: Helix 330-338 of factor IX interacts with residues 558-565 and spatially adjacent regions of the A2 subunit of factor VIIIa
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DOI：
10.1074/jbc.m011680200
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发表时间：
2001-05-11
影响因子：
4.8
通讯作者：
Fay, PJ
中科院分区：
文献类型：
--
作者：
Bajaj, SP;Schmidt, AE;Fay, PJ
因子X的生理激活剂由因子IXa、因子VIIIa、Ca 2+和合适的磷脂表面的复合物组成。在一项研究中，因子IXa蛋白酶结构域的螺旋330（胰凝乳蛋白酶中为162）与因子VIIIa结合有关。在另一项研究中，因子VIIIa的A2亚基的残基558-565涉及与因子IXa的结合。我们现在提供的数据表明，因子IXa的螺旋330与A2亚基的558-565区域相互作用，因此，分离的A2亚基在增强因子X激活中的能力被IXa（R333 Q）和螺旋置换突变体严重削弱（IXa（helixVII），其中螺旋330-338被因子VII的结构域替换），但对于表皮生长因子1替换突变体（IXa（PCEGF 1），其中表皮生长因子1结构域被蛋白C的结构域替换），这是正常的。此外，根据在tenase测定中其抑制野生型IXa的能力和根据其与A2亚基结合时丹磺酰基荧光发射信号的变化，确定每个5-二甲基氨基萘-1-磺酰基（丹磺酰基）-Glu-Gly-Arg-IXa（dEGR-IXa）与A2亚基的亲和力。表观Kd（A2）值为：dEGR-IXa（WT）或dEGR-IXa（PCEGF 1）类似于100 nM，dEGR-IXa（R333 Q）类似于1.8 μ M，dEGR-IXa（helixVII）>10 μ M。在另外的实验中，我们测量了这些因子IXa分子对包含A2亚基的残基558-565的肽的亲和力。表观K-d（肽）值为：dEGR-IXa（WT）或dEGR-IXa（PCEGF 1）类似于4 μ M，dEGR-IXa（R333 Q）类似于62 μ M。因此，与野生型或PCEGF 1突变体相比，R333 Q突变体对A2亚基或A2 558-565肽的亲和力类似地降低。这些数据支持因子IXa的螺旋330与A2 558-565序列相互作用的结论。该信息用于模拟IXa蛋白酶结构域和A2亚基之间的界面，其也在本文中提供。
The physiologic activator of factor X consists of a complex of factor IXa, factor VIIIa, Ca2+ and a suitable phospholipid surface. In one study, helix 330 (162 in chymotrypsin) of the protease domain of factor IXa was implicated in binding to factor VIIIa. In another study, residues 558-565 of the A2 subunit of factor VIIIa were implicated in binding to factor IXa. We now provide data, which indicate that the helix 330 of factor IXa interacts with the 558-565 region of the A2 subunit, Thus, the ability of the isolated A2 subunit was severely impaired in potentiating factor X activation by IXa(R333Q) and by a helix replacement mutant (IXa(helixVII) in which helix 330-338 is replaced by that of factor VII) but it was normal for an epidermal growth factor 1 replacement mutant (IXa(PCEGF1) in which epidermal growth factor 1 domain is replaced by that of protein C). Further, affinity of each 5-dimethylaminonaphthalene-1-sulfonyl (dansyl)-Glu-Gly-Arg-IXa (dEGR-IXa) with the A2 subunit was determined from its ability to inhibit wild-type IXa in the tenase assay and from the changes in dansyl fluorescence emission signal upon its binding to the A2 subunit. Apparent K-d(A2) values are: dEGR-IXa(WT) or dEGR-IXa(PCEGF1) similar to 100 nM, dEGR-IXa(R333Q) similar to1.8 muM, and dEGR-IXa(helixVII) >10 muM. In additional experiments, we measured the affinities of these factor IXa molecules for a peptide comprising residues 558-565 of the A2 subunit, Apparent K-d(peptide) values are: dEGR-IXa(WT) or dEGR-IXa(PCEGF1) similar to4 muM, and dEGR-IXa(R333Q) similar to 62 muM. Thus as compared with the wild-type or PCEGF1 mutant, the affinity of the R333Q mutant for the A2 subunit or the A2 558-565 peptide is similarly reduced. These data support a conclusion that the helix 330 of factor IXa interacts with the A2 558-565 sequence. This information was used to model the interface between the IXa protease domain and the A2 subunit, which is also provided herein.