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-C1-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 (K2060, K2061, S2062, W2063, W2064) to be involved in membrane binding. Three double mutants were created, K2060Q-K2061Q, W2063Y-W2064Y and W2063A-W2064A, and expressed in a transient expression system. In addition, a FV variant in which all four residues were mutated, K2060Q-K2061Q-W2063A-W2064A, 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.
Nicolaes
G A
GA
The Wallenberg Laboratory, Department of Clinical Chemistry, University Hospital Malmö, Lund University, Sweden. Gerry.Nicolaes@klkemi.mas.lu.se
Villoutreix
B O
BO
Dahlbäck
B
B
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001665:Binding Sites; D015153:Blotting, Western; D019556:COS Cells / Q000737:chemistry; D015943:Factor Va / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D015951:Factor Xa / Q000187:drug effects; D006801:Humans; D008958:Models, Molecular; D020125:Mutation, Missense; D010314:Partial Thromboplastin Time; D010713:Phosphatidylcholines; D010718:Phosphatidylserines / Q000378:metabolism / Q000494:pharmacology; D010743:Phospholipids / Q000378:metabolism* / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D011517:Prothrombin Time; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D000911:Antibodies, Monoclonal; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D010743:Phospholipids; D011994:Recombinant Proteins; D015943:Factor Va; D015951:Factor Xa; C017251:1,2-oleoylphosphatidylcholine