Participation of γ-Carboxyglutamic Acid Residues in Metal Binding: 13C nmr Studies of The Interaction of Lanthanide Ions with Bovine Prothrombin Fragment (12-44)

Abstract

Comparative studies of Ca(II) binding to bovine prothrombin and abnormal prothrombin have led to the hypothesis that γ-carboxyglutamic acid (Gla) residues either participate directly in metal liganding or stabilize the tertiary structure of the protein to facilitate metal binding, or both. Extending previous studies of the binding of lanthanide ions to factor X, prothrombin and prothrombin fragment 1, the interaction of lanthanide ions with prothrombin fragment (12-44), which contains eight Gla residues, has been examined by natural abundanceC NMR spectroscopy at 67.88 mHz. The relationship between bound metal ions and carbon atoms in fragment (12-44) was determined using paramagnetic Pr(III) and Eu(III) based upon the strategy that the magnitude of the change in the chemical shift of resonances of the carbon atoms induced by bound metals is related to the interatomic distance between bound metal and carbon nuclei. Assignments of the resonances in the 13C spectrum of fragment (12-44) were based upon our studies of the chemical shifts of carbon resonances in DL-Gla and known chemical shifts of carbon resonances of the other amino acid residues. Titration of fragment (12-44) with Pr(III) or Eu(III) was accompanied by a downfield or upfield shift, respectively, of the γ-carboxyl carbon resonances and no shifts were observed for other carbon resonances. The shift of resonances within the γ-carboxyl carbon envelope appeared selective, suggesting the non-equivalence of the relationship of the γ-carboxyl group of the eight Gla residues to the bound metal ions. These data offer direct evidence that some, but perhaps not all, Gla residues participate in metal liganding.