FRET studies with Factor X mutants provide insight into the topography of the membrane-bound Factor X/Xa

FRET (fluorescence resonance energy transfer) studies have shown that the vitamin K-dependent coagulation proteases bind to membrane surfaces perpendicularly, positioning their active sites above the membrane surfaces. To investigate whether EGF (epidermal growth factor) domains of these proteases play a spacer function in this model of the membrane interaction, we used FRET to measure the distance between the donor fluorescein dye in the active sites of F1-FPR (fluorescein-D-Phe-Pro-Arg-chloromethane)-inhibited fXa (activated Factor Xa) and its N-terminal EGF deletion mutant (fXa-desEGF1), and the acceptor OR (octadecylrhodamine) dye incorporated into phospholipid vesicles composed of 80% phosphatidylcholine and 20 phosphatidylserine. The average distance of closest approach (L) between fluorescein in the active site and OR at the vesicle surface was determined to be 56 +/- angstrom (1 angstrom=0.1 nm) and 63 +/- 1 angstrom for fXa-desEGF1 compared with 72 +/- 2 angstrom and 75 +/- 1 angstrom for fXa, in the absence and presence of fVa (activated Factor V) respectively, assuming kappa(2) = 2/3. In comparison, an L value of 95 6 A was obtained for a S195C mutant of fXa in the absence of fVa in which fluorescein was attached directly to Cys(195) of fXa. These results suggest that (i) EGF1 plays a spacer function in holding the active site of fXa above the membrane surface, (ii) the average distance between fluorescein attached to F1-FPR in the active site of fXa and OR at the vesicle surface may not reflect the actual distance of the active-site residue relative to the membrane surface, and (iii) fVa alters the orientation and/or the height of residue 195 above the membrane surface.