CA2+-DEPENDENT INTERACTIONS BETWEEN GLA AND EGF DOMAINS IN HUMAN COAGULATION FACTOR-IX

The Ca2+-induced interaction between the Gla and EGF domains of human factor IX was investigated by means of three fragments: 6-kDa Gla, 19-kDa (EGF)2, and 25-kDa Gla-(EGF)2. Size-exclusion chromatography and spectroscopic measurements revealed that the Gla-EGF interaction is rather strong; it can be reconstituted by mixing the 6-kDa and 19-kDa fragments which form a stable 1:1 heterocomplex in the presence of Ca2+. By itself, the 6-kDa Gla self-associates in these conditions. The Gla-EGF interaction can be disrupted in 5 M urea where the compact structure of both domains is preserved. Binding of Ca2+ to 19-kDa (EGF)2 occurred with a K(d) of 71 muM in the absence and 108 muM in the presence of 5 M urea and stabilized the first EGF domain, increasing its T(m) by 12-degrees-C. Addition of Ca2+ to the 6-kDa and 25-kDa fragments produced biphasic changes in their fluorescence; the intensity increased slightly at low Ca2+ concentration and then decreased in a monotonic manner. In 5 M urea, only the decrease occurred, with apparent K(d)s of 0.33 and 0.30 mM for 6-kDa Gla and 25-kDa Gla-(EGF)2, respectively. Thus, in 5 M urea in the presence of Ca2+, the isolated Gla domain has a compact structure and Ca2+ binding properties similar to those in the 25-kDa fragment. In the absence of urea, the Gla domain interacts either with itself, when isolated, or with the first EGF domain when present, as in the 6-kDa/19-kDa heterocomplex, in the 25-kDa fragment and presumably intact factor IX.