Blood coagulation factor IX residues Glu78 and Arg94 provide a link between both epidermal growth factor-like domains that is crucial in the interaction with factor VIII light chain

Recently, we established that mutations at calcium-binding sites within the first epidermal growth factor (EGF)-like domain of activated factor IX affect its interaction with factor VIIIa (Lenting, P. J,, Christophe, O, D,, ter Maat, H,, Rees, D, J, G,, and Mertens, K, (1996) J, Biol, Chem. 271, 25332-25337), In the present study, we have investigated the functional role of residue Glu(78) which is not involved in calcium binding, Glu(78) is, also located in the first EGF like domain and, when mutated to Lys, is associated with severe hemophilia B. Because Glu(78) is conserved in related vitamin K-dependent proteins, it is difficult to understand how a mutation at this position is associated with factor M-specific function, In this study, we addressed the hypothesis that Glu(78) exerts its biological activity by interacting with another residue, One candidate was found to be the second EGF-like domain residue, Arg(94), which is also associated with severe hemophilia B when mutated, We constructed a series of mutants that included mutations at position 78 alone (Glu(78) to Lys/Glu(78) to Asp) or at both positions 78 and 94 (Glu(78) to Lys and Arg(94) to Asp), The functional parameters of immunopurified and activated mutants were compared with normal activated factor IX. Mutants were indistinguishable from normal factor Ma in cleaving the synthetic substrate CH3SO2-Leu-Gly-Arg-p-nitroanilide or activating factor X in the absence of factor VIIIa, In contrast, in the presence of factor VIIIa, factor Ma Glu(78) to Asp and factor Ma Glu(78) to Lys/Arg(94) to Asp were stimulated to the same extent as normal factor Ma, whereas factor Ma Glu(78) to Lys was markedly less stimulated (140-fold versus 2,000-fold), This suggests that residues 78 and 94 should carry an opposite charge for a normal interaction of factor Ma to factor VIIIa, This hypothesis was confirmed in inhibition studies employing synthetic peptides comprising the factor Ma-binding motifs of factor VIII heavy (Ser(558)-Gln(565)) Or light chain (Glu(1811)-Lys(1818)) and in direct binding studies, We propose that residues 78 and 94 link both EGF-like domains and thereby maintain the integrity of the factor VIII light chain binding site.