Prothrombin requires two sequential metal-dependent conformational transitions to bind phospholipid. Conformation-specific antibodies directed against the phospholipid-binding site on prothrombin.
Prothrombin is a gamma-carboxyglutamic acid-containing protein that binds to phospholipid vesicles in the presence of calcium ions after undergoing a metal ion-induced conformational transition. To integrate recent data into a scheme that is compatible with our knowledge of prothrombin-metal interaction, we have proposed a new model of prothrombin structure. In this model prothrombin undergoes two metal-dependent conformational transitions: PT----PT'----PT*. The first transition is not cation-specific, but the second transition is metal-selective for Ca(II), Sr(II), or Ba(II). Only the PT* conformer binds to phospholipid surfaces. To test this model, anti-prothrombin antibodies that only bind to prothrombin in the presence of Ca(II) but not Mg(II) (PT*-specific) were isolated, and termed anti-prothrombin X Ca(II)-specific. Half-maximal binding of antibody to prothrombin was observed at 0.1 mM CaCl2 or 1 mM SrCl2, but no binding was observed with Mg(II), Mn(II), or Ba(II). However, prothrombin in the presence of both Mg(II)/Ba(II) or Mn(II)/Ba(II) demonstrated significant interaction with the antibody. Prothrombin binding to phospholipid vesicles was inhibited by the anti-prothrombin X Ca(II)-specific antibody or its Fab fragment, but was not inhibited by anti-prothrombin X Mg(II) antibody or its Fab fragment directed at the PT' conformer. These results support this three-state model for prothrombin. The metal specificity characteristic of prothrombin-phospholipid interaction is a property required for the expression of the phospholipid-binding site in the binary prothrombin-metal complex.
D000906:Antibodies; D000936:Antigen-Antibody Complex; D001665:Binding Sites; D000939:Epitopes / Q000032:analysis; D006801:Humans; D007700:Kinetics; D008027:Light; D010743:Phospholipids / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011516:Prothrombin / Q000276:immunology / Q000378:metabolism*; D011863:Radioimmunoassay / Q000379:methods; D012542:Scattering, Radiation
D000906:Antibodies; D000936:Antigen-Antibody Complex; D000939:Epitopes; D010743:Phospholipids; D011516:Prothrombin