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 fVIIa⅐TF 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-GDfVIIa 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-fVIIa⅐TF and OR at the vesicle surface was determined to be 78 Å ( 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 fVIIa⅐TF 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.
Overall structure and function of TF-FVlla (1,18,19); it confers the ability to bind reversibly to anionic phospholipids. Next to the Gla-domain, and sometimes classified as part of it, is a short shetch of largely hydrophobic residues called the hydrophobic or aromatic stack. Next are two modules related to epidermal growth factor (EGF domains). EGFr, the more N-terminal of the two, contains a
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