Hydrophobic contact between the two epidermal growth factor-like domains of blood coagulation factor IX contributes to enzymatic activity

The three-dimensional structure of activated factor IX comprises multiple c ontacts between the two epidermal growth factor (EGF)-like domains. One of these is a salt bridge between Glu(78) and Arg(94), which is essential for binding of factor Ma to its cofactor factor VIII and for factor VIII-depend ent factor X activation (Christophe, O. D., Lenting, P. J., Kolkman, J. A. Brownlee, G. G., and Mertens, K. (1998) J. Biol. Chem. 273, 222-227). We no w addressed the putative hydrophobic contact at the interface between the E GF-like domains, Recombinant factor IX chimeras were constructed in which h ydrophobic regions Phe(75)-Phe(77) and Lys(106)-Val(108) were replaced by t he corresponding sites of factor X and factor VII, Activated factor IX/fact or X chimeras were indistinguishable from normal factor Ma with respect to factor Ma enzymatic activity. In contrast, factor IXa(75-77)/factor VII dis played similar to 2-fold increased factor X activation in the presence of f actor VIII, suggesting that residues 75-77 contribute to cofactor-dependent factor X activation. Activation of factor X by factor IX106-108/factor VII was strongly decreased, both in the absence and presence of factor VIII. A ctivity could be restored by simultaneous substitution of the hydrophobic s ites in both EGF-like domains for factor VII residues. These data suggest t hat factor Ma enzymatic activity requires hydrophobic contact between the t wo EGF-like domains.