Antibody-probed conformational transitions in the protease domain of human factor IX upon calcium binding and zymogen activation: putative high-affinity Ca(2+)-binding site in the protease domain.
Antibody-probed conformational transitions in the protease domain of human factor IX upon calcium binding and zymogen activation: putative high-affinity Ca(2+)-binding site in the protease domain.
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钙结合和酶原激活时人因子 IX 蛋白酶结构域中抗体探测的构象转变：蛋白酶结构域中推定的高亲和力 Ca(2 ) 结合位点。
DOI：
10.1073/pnas.89.1.152
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发表时间：
1992
影响因子：
11.1
通讯作者：
Birktoft,JJ
中科院分区：
文献类型：
--
作者：
Bajaj,SP;Sabharwal,AK;Gorka,J;Birktoft,JJ
与人因子IX的蛋白酶结构域的N-末端一半（残基180-310）反应的单克隆抗体（mAb）的Fab片段先前已显示抑制因子IXa与其辅因子VIIIa的结合。这些数据表明，这部分的因子IXa可能参与结合因子VIIIa。我们现在报告的mAb的结合率（kon）是3倍，在Ca 2+的存在下，比在其缺乏的两个因子IX和IXa;在约300 μ M的Ca 2+观察到的半最大效应。此外，mAb对因子IXa的解离速率（koff）比有或无Ca 2+的因子IX高10倍。此外，与mAb结合的kon类似，在存在Ca 2+的情况下，丹磺酰-Glu-Gly-Arg氯甲基酮（dEGR-CK）掺入因子IXa的速度比不存在Ca 2+的情况快约3倍。由于空间因子支配的kon和非共价相互作用的强度支配的koff，数据表明，在残基180-310的因子IX的区域经历了两个独立的构象变化之前，其生物活性的表达：一个后，Ca 2+结合和其他酶原激活。此外，dEGF-CK掺入数据表明，这两种构象变化也影响活性位点残基。对丝氨酸蛋白酶已知三维结构的分析表明，在人因子IX中，谷氨酸235和245的羧基以及残基237和240的主链羰基氧可能形成高亲和力的Ca（2+）结合位点。为了支持这一结论，包括残基231-265的合成肽显示以约500 μ M的Kd结合Ca 2+。该肽也与mAb结合，尽管亲和力降低了约500倍。此外，与因子IX一样，在Ca 2+存在下，肽与mAb的结合比不存在时更强（约3倍）。因此，似乎上述mAb的表位的一部分包含在人因子IX的蛋白酶结构域中提出的Ca（2+）结合位点中。该位点类似于胰蛋白酶和弹性蛋白酶中的Ca（2+）结合位点，可能参与因子IXa与因子VIIIa的结合。
The Fab fragment of a monoclonal antibody (mAb) reactive to the N-terminal half (residues 180-310) of the protease domain of human factor IX has been previously shown to inhibit the binding of factor IXa to its cofactor, factor VIIIa. These data suggested that this segment of factor IXa may participate in binding to factor VIIIa. We now report that the binding rate (kon) of the mAb is 3-fold higher in the presence of Ca2+ than in its absence for both factors IX and IXa; the half-maximal effect was observed at approximately 300 microM Ca2+. Furthermore, the off rate (koff) of the mAb is 10-fold higher for factor IXa than for factor IX with or without Ca2+. Moreover, like the kon for mAb binding, the incorporation of dansyl-Glu-Gly-Arg chloromethyl ketone (dEGR-CK) into factor IXa was approximately 3 times faster in the presence of Ca2+ than in its absence. Since steric factors govern the kon and the strength of noncovalent interactions governs the koff, the data indicate that the region of factor IX at residues 180-310 undergoes two separate conformational changes before expression of its biologic activity: one upon Ca2+ binding and the other upon zymogen activation. Furthermore, the dEGF-CK incorporation data suggest that both conformational changes also affect the active site residues. Analyses of the known three-dimensional structures of serine proteases indicate that in human factor IX a high-affinity Ca(2+)-binding site may be formed by the carboxyl groups of glutamates 235 and 245 and by the main chain carbonyl oxygens of residues 237 and 240. In support of this conclusion, a synthetic peptide including residues 231-265 was shown to bind Ca2+ with a Kd of approximately 500 microM. This peptide also bound to the mAb, although with approximately 500-fold reduced affinity. Moreover, like factor IX, the peptide bound to the mAb more strongly (approximately 3-fold) in the presence of Ca2+ than in its absence. Thus, it appears that a part of the epitope for the mAb described above is contained in the proposed Ca(2+)-binding site in the protease domain of human factor IX. This proposed site is analogous to the Ca(2+)-binding site in trypsin and elastase, and it may be involved in binding factor IXa to factor VIIIa.