The factor V C1 domain is involved in membrane binding: identification of functionally important amino acid residues within the C1 domain of factor V using alanine scanning mutagenesis.
The contribution of the factor Va C1 domain (fVa-C1) to assembly of the prothrombinase complex has not been previously investigated. The homologous fVa-C2 domain contains a binding site for phosphatidylserine (PS) that includes the indole moieties of Trp(2063)/Trp(2064) at the apex of spike-1. In order to investigate the structure and function of fVa-C1 a molecular model was constructed based on the structure of fVa-C2. The aromatic and hydrophobic side chains of Tyr (1956) /Leu (1957) in fVa-C1 are located at the predicted apex of spike-3. Exposed charged and hydrophobic residues in fVa-C1 were changed to alanine in clusters of 1-3 mutations per construct. The resultant 20 mutants were expressed in COS cells and screened for binding to immobilized PS and prothrombinase activity on phospholipid vesicles containing either 25% or 5% PS. Two mutants, (Y1956,L1957)A, and (R2023,R2027)A showed both decreased binding to immobilized PS and a selective decrease in prothrombinase activity on membranes containing 5% PS. The interaction of purified (Y1956,L1957)A with phospholipid vesicles was studied using fluorescence resonance energy transfer and prothrombinase assays. The affinity of (Y1956,L1957)A binding to 25% PS membranes was reduced 12-fold compared to rHFVa. Prothrombin activation in the presence of (Y1956,L1957)A was markedly impaired on phos-pholipid vesicles containing 10% or less PS. We conclude that solvent exposed hydrophobic and aromatic amino acids in both fVa-C1 and fVa-C2 contribute to the interaction of factor V with PS membranes.
Saleh
Mahasen
M
Division of Hematology, Department of Medicine, DUMC-3656, Duke University Medical Center Durham, NC 27710, USA.
Peng
Weimin
W
Quinn-Allen
Mary Ann
MA
Macedo-Ribeiro
Sandra
S
Fuentes-Prior
Pablo
P
Bode
Wolfram
W
Kane
William H
WH
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism*; D017077:Culture Media, Conditioned / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005165:Factor V / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010718:Phosphatidylserines / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013925:Thromboplastin / Q000737:chemistry / Q000378:metabolism
D017077:Culture Media, Conditioned; D018076:DNA, Complementary; D010718:Phosphatidylserines; D010743:Phospholipids; D005165:Factor V; D013925:Thromboplastin; D000409:Alanine