Derivatives of blood coagulation factor IX contain a high affinity Ca2+-binding site that lacks gamma-carboxyglutamic acid.

We have examined the calcium-binding properties and metal ion-dependent conformational changes of proteolytically modified derivatives of factor IX that lack gamma-carboxyglutamic acid (Gla) residues. Equilibrium dialysis experiments demonstrated that a Gla-domainless factor IX species retained a single high affinity calcium ion-binding site (Kd = 85 +/- 5 microM). Ca2+ binding to this site was accompanied by a decrease in intrinsic fluorescence emission intensity (Kd = 63 +/- 15 microM). These spectral changes were reversed upon the addition of EDTA. Titration with Sr2+ resulted in little change in fluorescence intensity below 1 mM, while titration with Tb3+ caused fluorescence changes similar to those observed with Ca2+. Tb3+ and Ca2+ appear to bind to the same site because tryptophan-dependent terbium emission was reduced by the addition of Ca2+. Similar results were obtained with a Gla-domainless factor IX species lacking the activation peptide. Gla domain-containing factor IX species exhibited fluorescence changes similar to those of the Gla-domainless proteins at low Ca2+, but an additional structural transition was found at higher Ca2+ concentrations (apparent Kd greater than 0.8 mM). Thus, the conformations of factor IX proteins are nucleated and/or stabilized by calcium binding to a high affinity site which does not contain Gla residues. The binding of Ca2+ to lower affinity Gla domain-dependent metal ion-binding sites elicits an additional conformational change. The strong similarities between these results and those obtained with protein C (Johnson, A. E., Esmon, N. L., Laue, T. M. & Esmon, C. T. (1983) J. Biol. Chem. 258, 5554-5560), coupled with the remarkable sequence homologies of the vitamin K-dependent proteins, suggest that the high affinity Gla-independent Ca2+-binding site may be a common feature of vitamin K-dependent proteins.