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 Ca 2+ ‐binding experiments have indicated that the EGF‐module region of bovine protein S harbors high‐affinity Ca 2+ ‐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 β‐hydroxylation. Both modules were folded to a native conformation, as judged by immunochemical experiments and NMR spectroscopy. Ca 2+ binding to the modules was monitored with 1 H‐NMR spectroscopy. At physiological pH and 0.15 M NaCl, each module was found to have a single Ca 2+ ‐binding site with low affinity, i.e. K d values of 6.1 mM for the third and 8.6 mM for the fourth EGF module. At low salt conditions the Ca 2+ affinities are 5.2 mM and 0.6 mM, respectively. This Ca 2+ affinity is similar to that of the isolated N‐terminal EGF module from coagulation factors IX and X. The very high affinity Ca 2+ binding to the EGF‐module region of protein S thus appears to be due to the influence of neighboring modules.