Mutations in a potential phospholipid binding loop in the C2 domain of factor V affecting the assembly of the prothrombinase complex

Activated factor V (FVa) serves as a cofactor to activated factor X in the prothrombinase complex. FVa is homologous to activated factor VIII (FVIIIa), the light chains of both proteins being formed by similar domains (A3-CI-C2). Interaction of FVa and FVIIIa with negatively charged phospholipid membranes is crucial for the function of both cofactors. In both proteins, the C2 domains are important for membrane binding but a detailed understanding of the binding mechanisms is missing. Recently, knowledge has been gained into the three-dimensional structures of the C domains facilitating studies of structure-function relationships. Structural analysis of the C2 domain in FVa predicted a surface-exposed loop (K-2060, K-2061, S-2062, W-2063, W-2064) to be involved in membrane binding. Three double mutants were created, K(2060)Q-K(2061)Q, (WY)-Y-2063-(WY)-Y-2064 and W(2063)A-W(2064)A, and expressed in a transient expression system. In addition, a FV variant in which all four residues were mutated, K(2060)Q-K-2061 Q-W(2063)A-W(2064)A, was produced. Mutagenesis of the two lysines showed no functional consequences, whereas mutagenesis of the two tryptophanes yielded FVa with impaired ability to interact with the phospholipid, as demonstrated by a poor functional activity at limiting phospholipid concentrations. A molecular model of FVa, anchored at the surface of a phospholipid membrane, was developed and used as a template for the interpretation of the mutagenesis experiments. (C) 2000 Lippincott Williams & Wilkins.