title: Ca(2+)-dependent interactions between Gla and EGF domains in human coagulation factor IX.
abstract: The Ca(2+)-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 Kd of 71 microM in the absence and 108 microM in the presence of 5 M urea and stabilized the first EGF domain, increasing its Tm 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 Kds 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.
authors.affiliation: J. Holland Laboratory, American Red Cross, Rockville, Maryland 20855.
authors.forename: L V
authors.initials: LV
authors.lastname: Medved
authors.forename: A
authors.initials: A
authors.lastname: Vysotchin
authors.forename: K C
authors.initials: KC
authors.lastname: Ingham
mesh_terms: D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D002850:Chromatography, Gel; D004815:Epidermal Growth Factor / Q000737:chemistry*; D005164:Factor IX / Q000737:chemistry* / Q000378:metabolism; D005971:Glutamates / Q000737:chemistry*; D018698:Glutamic Acid; D006358:Hot Temperature; D006801:Humans; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry*; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D014508:Urea / Q000494:pharmacology
chemical_list: D005971:Glutamates; D010446:Peptide Fragments; D018698:Glutamic Acid; D004815:Epidermal Growth Factor; D014508:Urea; D005164:Factor IX; D002118:Calcium