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 modules, all of which have one partially hydroxylated Asp (EGF 1; beta-hydroxyaspartic acid) or Asn (EGF 2, 3, and 4; beta-hydroxyasparagine) residue. The three C-terminal modules have a typical Ca2+ binding sequence motif that is usually present in EGF modules with hydroxylated Asp/Asn residues. Using the chromophoric Ca2+ chelators Quin 2 and 5,5'-Br2BAPTA, we have now determined the Ca2+ affinity of recombinant fragments containing EGF modules 1-3, 1-4, 2-3, and 2-4. EGF modules 1-4 and 2-4 each contains two very high affinity Ca2+-binding sites, i.e. with dissociation constants ranging from 10(-10) to 10(-8) M in the absence of salt and from 10(-8) to 10(-6) M in the presence of 0.15 M NaCl. In contrast, in EGF 1-3 and EGF 2-3, the Ca2+ affinity is 2-4 orders of magnitude lower. EGF 4 thus appears to have the highest Ca2+ affinity, and furthermore it seems to influence the Ca2+ affinity of its immediate 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 factor 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 with neighboring modules. This difference is much higher than the approximately 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.
Stenberg
Y
Y
Department of Clinical Chemistry, University of Lund, University Hospital, Malmö, Sweden.
Linse
S
S
Drakenberg
T
T
Stenflo
J
J
D000595:Amino Acid Sequence; D000634:Aminoquinolines; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002614:Chelating Agents; D004533:Egtazic Acid / Q000031:analogs & derivatives; D004815:Epidermal Growth Factor / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D017293:Protein S / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera / Q000166:cytology / Q000821:virology; D017186:Titrimetry
D000634:Aminoquinolines; D002135:Calcium-Binding Proteins; D002614:Chelating Agents; D010446:Peptide Fragments; D017293:Protein S; D011994:Recombinant Proteins; C053459:5,5'-dibromo-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D004533:Egtazic Acid; D004815:Epidermal Growth Factor; C035739:Quin2; D002118:Calcium