THE HIGH-AFFINITY CALCIUM-BINDING SITES IN THE EPIDERMAL GROWTH-FACTOR MODULE REGION OF VITAMIN-K-DEPENDENT PROTEIN-S

Vitamin K-dependent protein S, a cofactor of the anticoagulant enzyme- activated protein C, has four epidermal growth factor (EGF)-like modul es, all of which have one partially hydroxylated Asp (EGF 1; beta-hydr oxyaspartic acid) or Asn (EGF 2, 3, and 4; beta-hydroxyasparagine) res idue, The three C-terminal modules have a typical Ca2+ binding sequenc e motif that is usually present in EGF modules with hydroxylated Asp/A sn residues, Using the chromophoric Ca2+ chelators Quin 2 and 5,5'-Br( 2)BAPTA, we have now determined the Ca2+ affinity of recombinant fragm ents containing EGF modules 1-3, 1-4, 2-3, and 2-4. EGF modules 1-4 an d 2-4 each contains two very high affinity Ca2+-binding sites, i.e. wi th dissociation constants ranging from 10(-10) to 10(-8) M in the abse nce of salt and from 10(-8) to 10(-6) M in the presence of 0.15 M NaCl . In contrast, in EGF 13 and EGF 2-3, the Ca2+ affinity is 2-4 orders of magnitude lower, EGF 4 thus appears to have the highest Ca2+ affini ty, and furthermore it seems to influence the Ca2+ affinity of its imm ediate N-terminal neighbor EGF 3 by a factor of approximately 230, In addition, EGF 4 seems to influence the Ca2+ affinity of EGF 2 by a fac tor of approximately 25, The Ca2+ affinity of the binding sites in EGF modules 3 and 4 in fragments EGF 1-4 and EGF 2-4 is 10(3)-10(5)-fold higher than in the corresponding isolated modules, implying important contributions to the Ca2+ affinity of each module from interactions wi th neighboring modules, This difference is much higher than the approx imately 10-fold difference previously found in similar comparisons of EGF modules from fibrillin. However, the modules studied in protein S and fibrillin appear to have the similar Ca2+ ligands, The structural basis for the difference in Ca2+ affinity is not yet understood.