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 Fl-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+/-1 A (1 A=0.1 nm) and 63+/-1 A for fXa-desEGF1 compared with 72+/-2 A and 75+/-1 A for fXa, in the absence and presence of fVa (activated Factor V) respectively, assuming kappa2=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 Fl-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.
Qureshi
Shabir H
SH
Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, MO 63104, USA.
Yang
Likui
L
Yegneswaran
Subramanian
S
Rezaie
Alireza R
AR
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000235:genetics; D015951:Factor Xa / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D017384:Sequence Deletion / Q000235:genetics
D008565:Membrane Proteins; D015951:Factor Xa