The N-terminal EGF domain of coagulation factor IX: Probing its functions in the activation of factor IX and factor X with a monoclonal antibody.

SUMMARY Absence or reduced activity of coagulation factor IX (FIX) causes the severe bleeding disorder hemophilia B. FIX contains an N-terminal Gla domain followed by two epidermal growth factor (EGF)-like domains and a serine protease domain. In this study the epitope of monoclonal antibody AW, which is directed against the C-terminal part of the first EGF domain in human FIX, was defined and the antibody was used to study interactions between the EGF domain of FIX and other coagulation proteins. Antibody AW completely blocks activation of FIX by FXIa, but activation by FVIIa/tissue factor is inhibited only slightly. The antibody also causes a marginal reduction in the apparent kcat for FX both in the presence and absence of FVIIIa. Based on these results, we produced a preliminary model of the structure of the FIXa-FVIIIa-AW complex on the surface of phospholipid. The model suggests that in the Xase complex EGF1 of FIXa is not involved in direct binding to FVIIIa. Studies of the interaction of antibody AW with a mutated FIX molecule (Arg94Asp) also suggest that the Glu78-Arg94 salt-bridge is not important for maintaining the structure of FIX

Solution Structure of the Ca(2+)-Binding EGF3-4 Pair from Vitamin K-Dependent Protein S: Identification of an Unusual Fold in EGF3(,).

Vitamin K-dependent protein S is a cofactor of activated protein C, a serine protease that regulates blood coagulation. Deficiency of protein S can cause venous thrombosis. Protein S has four EGF domains in tandem; domains 2-4 bind calcium with high affinity whereas domains 1-2 mediate interaction with activated protein C. We have now solved the solution structure of the EGF3-4 fragment of protein S. The linker between the two domains is similar to what has been observed in other calcium-binding EGF domains where it provides an extended conformation. Interestingly, a disagreement between NOE and RDC data revealed a conformational heterogeneity within EGF3 due to a hinge-like motion around Glu186 in the Cys-Glu-Cys sequence, the only point in the domain where flexibility is allowed. The dominant, bent conformation of EGF3 in the pair has no precedent among calcium-binding EGF domains. It is characterized by a change in the angle of Glu186 from 160 ± 40, as seen in ten other EGF domains, to 0 ± 15. NOESY data suggest that Tyr193, a residue not conserved in other calcium-binding EGF domains (except in the homologue Gas6), induces the unique fold of EGF3. However, SAXS data, obtained on EGF1-4 and EGF2-4, showed a dominant, extended conformation in these fragments. This may be due to a counterproductive domain-domain interaction between EGF2 and EGF4 if EGF3 is in a bent conformation. We speculate that the ability of EGF3 to adopt different conformations may be of functional significance in protein-protein interactions involving protein S