MEMBRANE-BINDING PEPTIDE FROM THE C2 DOMAIN OF FACTOR-VIII FORMS AN AMPHIPATHIC STRUCTURE AS DETERMINED BY NMR-SPECTROSCOPY

Factor VIII binds to cell membranes prior to assembling with the serin e protease, factor IXa, to form the factor X-activating enzyme complex . In order to better understand the interaction between factor VIII an d phosphatidylserine-containing membranes, we have synthesized the mem brane-binding peptide from the C2 domain of factor VIII, corresponding to residues 2303-2324. The peptide, fVIII(2303-24), with a primary st ructure of TRYLRIHPQSWVHQIALRMEVL, aggregates at concentrations above 2 mu M at pH 7 but is soluble at pH 6. fVIII(2303-24) competes with fl uorescein-labeled factor VIII (K-i = 3 mu M) for binding sites on synt hetic phosphatidylserine-containing membranes and for binding sites on stimulated platelets. Circular dichroism spectra indicate that fVIII( 2303-24) is predominantly a random coil in aqueous solution but adopts a predominantly helical conformation upon interaction with SDS micell es. H-1 NMR spectroscopy in the presence of SDS micelles allowed estim ation of interproton distances from the nuclear Overhauser effect and estimation of torsion angles from coupling constants indicated by spli tting of resonance lines. The distance and angle estimates, processed by distance geometry/simulated annealing software, indicate that fVIII (2303-24) has an alpha-helical segment encompassing residues P8-E20 an d an extended segment encompassing residues L4-P8. The location of six hydrophobic residues on one face of the structure suggests that hydro phobic interactions contribute to membrane-binding. In addition, two a rginines penetrate the hydrophobic plane suggesting that they interact with phosphate moieties in a phospholipid bilayer.