In addition to γ-carboxyglutamic acid (Gla)-dependent calcium binding all of the vitamin K-dependent plasma proteins, except prothrombin, have one or two high affinity calcium binding sites that do not require the Gla residues. A common denominator among these proteins (factors IX, X, protein C, protein Z and protein S) is that they have domaines that are homologus to the epidermal growth factor (EGF) precursor. In factors VII,IX,X, protein C and in protein Z the aminoterminal of two EGF homology regions contain one residue of β-hydroxyaspartic acid (Hya) whereas in protein S the aminoterminal EGF homology region contains Hya and the three following contain one β-hydroxyasparagine residue each.In an attempt to elucidate the role of the EGF homology regions in the Gla independent calcium binding we have isolated a tryptic fragment (residue 44-138) from the light chain of human protein C. The fragment was isolated using a monoclonal antibody that recognizes a calcium ion stabilized epitope that is expressed both in intact protein C and in protein C lacking the Gla domaine.The antibody bound the isolated EGF homology region in the presence of calcium ions but not in EDTA containing buffer. A calcium ion titration showed half maximal binding at approximately 200 μM Ca2+. The metal ion induced conformational change in the isolated fragment was also studied with affinity purified rabbit antibodies against Gla domainless protein C. Antibodies that bound in the presence of calcium ions and that could be eluted with EDTA recognized the metal ion induced conformational change in the isolated EGF homology domain. Our results suggest that one or both of the EGF homology regions are involved in the Gla-independent high affinity calcium binding in the vitamin K-dependent plasma proteins.
Structural changes induced by calcium and magnesium in a high affinity calcium-binding protein from crayfish sarcoplasm.