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 mem branes 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 exp ressed 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 p roduced. Mutagenesis of the two lysines showed no functional consequences, whereas mutagenesis of the two tryptophanes yielded FVa with impaired abili ty 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) 2 000 Lippincott Williams & Wilkins.