CALCIUM AFFINITY OF THE NH2-TERMINAL EPIDERMAL GROWTH FACTOR-LIKE MODULE OF FACTOR-X - EFFECT OF THE GAMMA-CARBOXYGLUTAMIC ACID-CONTAINING MODULE

The NH2-terminal epidermal growth factor (EGF)-like module of vitamin K-dependent coagulation factors IX and X and protein C each has one ca lcium binding site. This module (residues 45-86) from factor X has bee n isolated previously and found to bind calcium with a K(d) of 2.2 mM at physiological pH and ionic strength. We have now demonstrated that it binds calcium with a K(d) of 120 muM in a fragment that consists of the Gla module and the NH2-terminal EGF-like module. The presence of the Gla module (residues 1-44) increases the calcium affinity of the s ite in the EGF-like module approximately 20-fold, thus making it essen tially saturated in vivo. Decarboxylation of the Gla residues to Glu h as no significant effect on the calcium affinity of the EGF-like modul e. A proteolytic fragment of factor X (residues 29-86) and a synthetic peptide (residues 34-86), folded to a native conformation, were used to demonstrate that the contribution of the Gla module to the calcium affinity of the site in the EGF-like module is mediated by its 17 COOH -terminal residues, 12 of which form an alpha-helix in the intact Gla module. In the NMR structure of the NH2-terminal EGF-like module in fa ctor X, five calcium ligating groups have been identified (Selander-Su nnerhagen, M., Ullner, M., Persson, E., Teleman, O., Stenflo, J., and Drakenberg, T. (1992) J. Biol. Chem. 267, 19642-19649). As calcium usu ally requires seven to eight oxygen ligands, there is reason to believ e that the Gla module contributes ligands, or negative charge, to incr ease the calcium affinity. Our findings suggest that the calcium affin ity of EGF-like modules in other proteins may also be influenced by ne ighboring modules.