Calcium-binding properties of the third and fourth epidermal-growth-factor-like modules in vitamin-K-dependent protein S.
Protein S is a plasma glycoprotein requiring vitamin K for normal biosynthesis and functioning as a cofactor of activated protein C, a regulator of blood coagulation. Protein S contains four modules that are similar to the epidermal growth factor (EGF) precursor. Qualitative Ca2+-binding experiments have indicated that the EGF-module region of bovine protein S harbors high-affinity Ca2+-binding sites. We have chemically synthesized the third and fourth EGF modules from human protein S, which both have the sequence motif associated with Ca2+-binding and Asp/Asn beta-hydroxylation. Both modules were folded to a native conformation, as judged by immunochemical experiments and NMR spectroscopy. Ca2+ binding to the modules was monitored with 1H-NMR spectroscopy. At physiological pH and 0.15 M NaCl, each module was found to have a single Ca2+-binding site with low affinity, i.e. Kd values of 6.1 mM for the third and 8.6 mM for the fourth EGF module. At low salt conditions the Ca2+ affinities are 5.2 mM and 0.6 mM, respectively. This Ca2+ affinity is similar to that of the isolated N-terminal EGF module from coagulation factors IX and X. The very high affinity Ca2+ binding to the EGF-module region of protein S thus appears to be due to the influence of neighboring modules.
Stenberg
Y
Y
Department of Clinical Chemistry, University of Lund, University Hospital, Malmö, Sweden.
Julenius
K
K
Dahlqvist
I
I
Drakenberg
T
T
Stenflo
J
J
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D004815:Epidermal Growth Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D017293:Protein S / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014812:Vitamin K / Q000378:metabolism
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D017293:Protein S; D014812:Vitamin K; D004815:Epidermal Growth Factor; D002118:Calcium