Novel type of very high affinity calcium-binding sites in beta-hydroxyasparagine-containing epidermal growth factor-like domains in vitamin K-dependent protein S.
Vitamin K-dependent protein S is shown to contain four very high affinity Ca2(+)-binding sites. The number of sites and their affinities were determined from Ca2+ titration in the presence of the chromophoric chelator Quin 2. In 0.15 M NaCl, pH 7.5, the four macroscopic binding constants are K1 greater than or equal to 1 x 10(8) M-1, K2 = 3 +/- 2 x 10(7) M-1, K3 = 4 +/- 2 x 10(6) M-1, and K4 = 9 +/- 4 x 10(5) M-1. At low ionic strength, the corresponding values are K1 greater than or equal to 2 x 10(9) M-1, K2 = 9 +/- 4 x 10(8) M-1, K3 = 2 +/- 1 x 10(8) M-1, and K4 = 9 +/- 4 x 10(7) M-1. To localize the Ca2(+)-binding sites, protein S was subjected to proteolysis using lysyl endopeptidase. This yielded a 20-21-kDa fragment which comprised the third and fourth epidermal growth factor (EGF)-like domains and remained high affinity Ca2(+)-binding site(s). The susceptibility of the EGF-like domains to proteolysis increased when Ca2+ was removed from protein S indicating that the Ca2+ binding is important for the stability and/or conformation of the EGF domains. Three of the four EGF-like domains in protein S contain beta-hydroxyasparagine. In each of these domains there is a cluster of three or four negatively charged amino acid residues which are likely to contribute to the extraordinary high Ca2+ affinity. From sequence homology it is suggested that this novel type of high affinity Ca2(+)-binding site is present in several other proteins, e.g. in the EGF-like domains in the low sensity lipoproteins receptor, thrombomodulin, the Notch protein of Drosophila melanogaster, and transforming growth factor beta 1-binding protein.
Dahlbäck
B
B
Department of Clinical Chemistry, University of Lund, Malmö General Hospital, Sweden.
Hildebrand
B
B
Linse
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000031:analogs & derivatives; D001665:Binding Sites; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000276:immunology / Q000378:metabolism* / Q000648:ultrastructure; D002417:Cattle; D004492:Edetic Acid / Q000494:pharmacology; D010450:Endopeptidases / Q000494:pharmacology; D006023:Glycoproteins / Q000276:immunology / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D017293:Protein S; D013329:Structure-Activity Relationship
D002135:Calcium-Binding Proteins; D006023:Glycoproteins; D010446:Peptide Fragments; D017293:Protein S; C050931:3-hydroxyasparagine; D001216:Asparagine; D004492:Edetic Acid; D010450:Endopeptidases; D002118:Calcium