Tissue factor positions and maintains the factor VIIa active site far above the membrane surface even in the absence of the factor VIIa Gla domain - A fluorescence resonance energy transfer study
Tissue factor positions and maintains the factor VIIa active site far above the membrane surface even in the absence of the factor VIIa Gla domain - A fluorescence resonance energy transfer study
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DOI：
10.1074/jbc.272.48.30160
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发表时间：
1997-11-28
影响因子：
4.8
通讯作者：
Johnson, AE
中科院分区：
文献类型：
--
作者：
McCallum, CD;Su, BX;Johnson, AE
凝血因子VIIa （fVIIa）是一种可溶性丝氨酸蛋白酶，只有与它的辅因子组织因子（TF）结合时才表现出充分的蛋白水解活性。这两种蛋白质都与膜相互作用；TF是一种完整的膜蛋白，而fVIIa通过其Gla结构域可逆地与磷脂表面结合。在这项研究中，我们研究了fVIIa活性位点在fVIIa中的位置。TF配合物是由fVIIa glass结构域决定的。将一种荧光素染料通过三肽链与缺乏Gla结构域的fVIIa活性位点（无Gla结构域的fVIIa， GD-fVIIa）共价结合，得到荧光素- D-Phe-Pro-Arg-GD-fVIIa （Fl-FPR-GD-fVIIa）。利用荧光素染料在GD-fVIIa活性位点与磷脂囊泡表面十八烷基罗丹明（octadecylrhodamine， OR）之间的荧光共振能量转移，确定GD-fVIIa活性位点相对于膜表面的位置。正如预期的那样，在磷脂酰胆碱/磷脂酰丝氨酸组成的囊泡中，Fl-FPR-GD-fVIIa和OR之间没有观察到能量转移（PC/PS, 4:1），因为fVIIa与磷脂结合需要Gla结构域。然而，当用PC或PC/PS囊泡滴定Fl-FPR-GD-fVIIa时，观察到能量转移。基于荧光共振能量转移对OR密度的依赖性，荧光素在Fl-FPR-GD-fVIIa活性位点的平均最接近距离。测定囊泡表面的TF和OR为78埃（kappa(2) = 2/3）。由于该值与完整的Fl-FPR-fVIIa结合TF得到的值几乎相同，因此fVIIa Gla结构域的存在与否对fVIIa活性位点的位置影响很小。TF复杂。因此，组织因子的细胞外结构域相对于表面必须是相当刚性和固定的，以便即使在没有fVIIa Gla结构域的情况下，也能将fVIIa活性位点定位并维持在远高于膜的位置。
Coagulation factor VIIa (fVIIa), a soluble serine protease, exhibits full proteolytic activity only when bound to its cofactor, tissue factor (TF). Both proteins interact with membranes; TF is an integral membrane protein, while fVIIa binds reversibly to phospholipid surfaces via its Gla domain. In this study, we examine the extent to which the location of the fVIIa active site in the fVIIa . TF complex is determined by the fVIIa Gla domain. A fluorescein dye was covalently attached to the active site of fVIIa lacking the Gla domain (Gla domainless fVIIa, GD-fVIIa) via a tripeptide tether to yield fluorescein- D-Phe-Pro-Arg-GD-fVIIa (Fl-FPR-GD-fVIIa). The location of the active site of GD-fVIIa relative to the membrane surface was determined using fluorescence resonance energy transfer between the fluorescein dye in the active site of GD-fVIIa and octadecylrhodamine (OR) at the surface of phospholipid vesicles. As expected, no energy transfer was observed between Fl-FPR-GD-fVIIa and OR in vesicles composed of phosphatidylcholine/phosphatidylserine (PC/PS, 4:1) because the Gla domain is required for the binding of fVIIa to phospholipid. However, when Fl-FPR-GD-fVIIa was titrated with PC or PC/PS vesicles into which purified TF had been reconstituted, energy transfer was observed. Based on the dependence of fluorescence resonance energy transfer on OR density, the average distance of closest approach between fluorescein in the active site of Fl-FPR-GD-fVIIa . TF and OR at the vesicle surface was determined to be 78 Angstrom (kappa(2) = 2/3). Since this value is nearly the same as that obtained with intact Fl-FPR-fVIIa bound to TF, the presence or absence of the fVIIa Gla domain has only a small effect on the location of the active site in the fVIIa . TF complex. The extracellular domain of tissue factor therefore must be fairly rigid and fixed relative to the surface to position and maintain the fVIIa active site far above the membrane even in the absence of the fVIIa Gla domain.