CALCIUM-BINDING PROPERTIES OF THE 3RD AND 4TH EPIDERMAL-GROWTH-FACTOR-LIKE MODULES IN VITAMIN-K-DEPENDENT PROTEIN-S

Protein S is a plasma glycoprotein requiring vitamin K for normal bios ynthesis and functioning as a cofactor of activated protein C, a regul ator of blood coagulation. Protein S contains four modules that are si milar to the epidermal growth factor (EGF) precursor. Qualitative Ca2-binding experiments have indicated that the EGF-module region of bovi ne protein S harbors high-affinity Ca2+-binding sites. We have chemica lly synthesized the third and fourth EGF modules from human protein S, which both have the sequence motif associated with Ca2+-binding and A sp/Asn beta-hydroxylation. Both modules were folded to a native confor mation, as judged by immunochemical experiments and NMR spectroscopy. Ca2+ binding to the modules was monitored with H-1-NMR spectroscopy. A t physiological pH and 0.15 M NaCl, each module was found to have a si ngle Ca2+-binding site with low affinity, i.e. K-d values of 6.1 mM fo r the third and 8.6 mM for the fourth EGF module. At low salt conditio ns the Ca2+ affinities are 5.2 mM and 0.6 mM, respectively. This Ca2affinity 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 influ ence of neighboring modules.