We introduce here a new model to describe the binding of extrinsic membrane proteins to acidic lipid membranes. In this view, macroscopic binding affinity is determined by two processes: nonspecific adsorption of protein to the membrane surface and association of acidic lipids with specific sites on the bound protein. We apply this model here to compare the binding of human prothrombin and factor X/Xa to phosphatidylglycerol (PG)- and phosphatidylserine (PS)-containing small unilamellar vesicles measured via relative light scattering. This comparison was undertaken because model membranes containing PS are much more effective in supporting thrombin formation than are membranes containing PG. Analysis of binding isotherms in terms of a traditional membrane binding model gave apparent dissociation constants systematically varying from 0.1 to 10 microM over a range of 8-65 mol% negatively charged phospholipid. With our new description of membrane binding, the dependence of binding data on the acidic lipid surface concentration revealed that only two or three acidic lipid molecules were associated with each surface-bound factor X/Xa or prothrombin molecule. Assuming four independent and equivalent acidic lipid binding sites per protein, it was possible to adjust the values of only the nonspecific adsorption equilibrium constant and the equilibrium constant describing binding of each species of acidic lipid to individual sites on the protein and thereby obtain a good simulation of log-linear binding isotherms for the full range of acidic lipid surface concentrations. The protein-associated binding sites had a greater affinity for PS than for PG; i.e., a lower surface concentration of PS was required to fill the binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

The mechanism of binding of blood coagulation cofactor factor Va to acidic-lipid-containing membranes has been addressed. Binding isotherms were generated at room temperature using the change in fluorescence anisotropy of pyrene-labeled bovine factor Va to detect binding to sonicated membrane vesicles containing either bovine brain phosphatidylserine (PS) or 1,2-dioleoyl-3-sn-phosphatidylglycerol (DOPG) in combination with 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine (POPC). The composition of the membranes was varied from 0 to 40 mol% for PS/POPC and from 0 to 65 mol % for DOPG/POPC membranes. Fitting the data to a classical Langmuir adsorption model yielded estimates of the dissociation constant (Kd) and the stoichiometry of binding. The values of Kd defined in this way displayed a maximum at low acidic lipid content but were nearly constant at intermediate to high fractions of acidic lipid. Fitting the binding isotherms to a two-process binding model (nonspecific adsorption in addition to binding of acidic lipids to sites on the protein) suggested a significant acidic-lipid-independent binding affinity in addition to occupancy of three protein sites that bind PS in preference to DOPG. Both analyses indicated that interaction of factor Va with an acidic-lipid-containing membrane is much more complex than those of factor Xa or prothrombin. Furthermore, a change in the conformation of bound pyrene-labeled factor Va with surface concentration of acidic lipid was implied by variation of both the saturating fluorescence anisotropy and the binding parameters with the acidic lipid content of the membrane. Finally, the results cannot support the contention that binding occurs through nonspecific adsorption to a patch or domain of acidic lipids in the membrane. Factor Va is suggested to associate with membranes by a complex process that includes both acidic-lipid-specific and acidic-lipid-independent sites and a protein structure change induced by occupancy of acidic-lipid-specific sites on the factor Va molecule.

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