Domain structure and domain-domain interactions in human coagulation factor IX.
Coagulation factor IX has the modular composition Gla-(EGF)2-SP, where Gla, EGF, and SP represent the gamma-carboxy-Glu-rich, epidermal growth factor-like, and serine protease modules, respectively. The protein melts in two distinct temperature regions. The SP module melts at lower temperature between 42 and 55 degrees C, depending on the pH, with irreversible loss of esterolytic activity. The endotherm corresponding to this transition is readily described by a two-state transition indicating the melting of a single cooperative unit. A thrombin-generated 12-kDa fragment representing the COOH-terminal half of the SP module and a 45-kDa fragment containing the NH2-terminal half of the SP module and the rest of the molecule can be separated by exclusion chromatography in 3 M urea and recombined in its absence. Both fragments retain a compact structure as evidenced by melting transitions near 60 degrees C at neutral pH. This indicates that the SP module contains two independently folded domains that strongly interact with each other and seem to merge into one cooperative unit in the intact protein. At low pH at high temperature a second peak appears which is also observed in a 19-kDa fragment containing the EGF modules. Thermodynamic analysis of this second peak showed that the two EGF modules are independently folded and provided evidence for a weak interaction between them. Fluorescence and CD measurements indicated that the secondary structure of the isolated 6-kDa Gla fragment is substantially increased in the presence of Ca2+. The Ca2+-loaded Gla fragment undergoes a sigmoidal unfolding transition as revealed by fluorescence and CD measurements. This same transition in a 25-kDa Gla-(EGF)2 fragment was stabilized by more than 10 degrees C, indicating a strong interaction between the Ca(2+)-loaded Gla and EGF domains. Thus, factor IX consists of five independently folded interacting domains.
Vysotchin
A
A
Holland Laboratory, American Red Cross, Rockville, Maryland 20855.
Medved
L V
LV
Ingham
K C
KC
D002152:Calorimetry, Differential Scanning; D005164:Factor IX / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D012697:Serine Endopeptidases / Q000378:metabolism; D013816:Thermodynamics
D010446:Peptide Fragments; D005164:Factor IX; D012697:Serine Endopeptidases