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Volume 271, Number 45, Issue of November 8, 1996 pp. 28168-28175
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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The Location of the Active Site of Blood Coagulation Factor VIIa above the Membrane Surface and Its Reorientation upon Association with Tissue Factor
A FLUORESCENCE ENERGY TRANSFER STUDY

(Received for publication, May 8, 1996, and in revised form, August 26, 1996)

Christine D. McCallum , Raymond C. Hapak ^§ , Pierre F. Neuenschwander , James H. Morrissey and Arthur E. Johnson ^§§

From the  Department of Medical Biochemistry & Genetics, Texas A&M University Health Science Center, College Station, Texas 77843-1114, the ^§ Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019, the  Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, and the ^§§ Departments of Chemistry and of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843

The topography of membrane-bound blood coagulation factor VIIa (fVIIa) was examined by positioning a fluorescein dye in the active site of fVIIa via a tripeptide tether to yield fluorescein-D-phenylalanyl-L-prolyl-L-arginyl-fVIIa (Fl-FPR-fVIIa). The location of the active-site probe relative to the membrane surface was determined, both in the presence and absence of tissue factor (TF), using fluorescence energy transfer between the fluorescein dye and octadecylrhodamine (OR) at the phospholipid vesicle surface. When Fl-FPR-fVIIa was titrated with phospholipid vesicles containing OR, the magnitude of OR-, calcium ion-, and phosphatidylserine-dependent fluorescence energy transfer revealed that the average distance of closest approach between fluorescein in the active site of fVIIa and OR at the vesicle surface is 82 Å assuming a random orientation of donor and acceptor dyes (² = 2/3; the orientational uncertainty totals ~10%). The active site of fVIIa is therefore located far above the membrane surface, and the elongated fVIIa molecule must bind at one end to the membrane and project approximately perpendicularly out of the membrane.

When Fl-FPR-fVIIa was titrated with vesicles that contained TF, the efficiency of energy transfer was increased by a TF-dependent translational and/or rotational movement of the fVIIa protease domain relative to the membrane surface. If this movement was solely translational, the height of the active site of fVIIa was lowered by an average of 6 Å after binding to TF. The association of fVIIa with TF on the membrane surface therefore causes a significant reorientation of the active site relative to the membrane surface. This cofactor-dependent realignment of the active-site groove presumably facilitates and optimizes fVIIa cleavage of its membrane-bound substrates.

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