The C2 domain epitope consists of two loops, Glu2181-Ala2188 and Thr2202-Arg2215 (Fig. of the immune response to factor VIII following hemophilia A treatment will help lead to the development of better therapeutic reagents. AP1867 Hemophilia A is a blood clotting disorder caused by a lack of functional blood coagulation factor VIII (fVIII), a protein cofactor essential to the intrinsic pathway of the blood clotting cascade. AP1867 Congenital hemophilia A, which varies in severity depending on the amount of functional fVIII present, is an X-linked disorder affecting 1 in 5,000 males worldwide1. The primary treatment for the disease is therapeutic infusions of recombinant fVIII, either in an acute or prophylactic manner2,3. The most significant complication to this treatment is the development of neutralizing inhibitory antibodies directed against the infused fVIII. Approximately 30% of patients receiving replacement therapy develop inhibitory antibodies, an immune response leading to the clearance of fVIII from circulation and continued lack of clotting function4,5,6. Coagulation fVIII is a 2,332-residue GYPA glycoprotein that is expressed with the domain arrangement of A1-A2-B-Clashscore12.62?Average B-factor (?2)49.4??macromolecules49.6??ligands70.1??solvent41.1PDB code4XZU Open in a separate window The overall structure of the fVIII C2 domain/3E6 FAB binding interface is highly conserved. Upon superposition of the C2 domain and variable domains for each binary complex with the C2/3E6 portion of the previously determined structure of the C2 domain/3E6/G99 FAB ternary complex28, the RMSD for complexes 1 and 2 were calculated to be 0.328 and 0.383 for C atoms, respectively (Fig. 1b). The most AP1867 significant deviation in the C2/3E6 binary crystal structure was present at the AP1867 elbow angle between the variable and constant domains of each complex. Specifically, the C2/3E6 portion of the ternary complex displayed the most extended structure, with an elbow angle close to zero. By contrast, complexes 1 and 2 possessed increasing deviations from planarity about the FAB elbow, respectively (Fig. 1b). While this discrepancy is notable, changes in elbow angles for FAB structures are often present and likely do not contribute significantly to the observed cooperativity for anti-C2 domain antibody binding36. To further understand the solution conformation of the 3E6 FAB in complex with the fVIII C2 domain, each C2/3E6 binary structure was fit into a newly calculated SAXS envelope of the C2/3E6 complex from previously collected SAXS data29. Subsequent to manual alignment of each structure with the SAXS envelope, the Fit in Map algorithm in Chimera was employed to optimize the alignment and calculate a correlation coefficient. While all the models fit within reason to the SAXS envelope, the C2/3E6 structures from binary complex 1 and the ternary complex yielded the highest correlation AP1867 ( 0.97), indicating that the solution conformation of the C2/3E6 complex is more extended with an FAB elbow angle approaching 180 (Fig. 2). Open in a separate window Figure 2 SAXS envelope of the factor VIII C2 domain/3E6 FAB complex. Based on previous SAXS data29, molecular envelopes were calculated with DAMMIF, averaged with DAMAVER and refined with DAMMIN.Rigid body modeling of the C2 domain/3E6 complex from the C2/3E6/G99 ternary structure28 was modeled as a rigid body into the SAXS envelope with the Fit in Map algorithm in Chimera. The factor VIII C2 domain/3E6 FAB binding interface The 3E6 antibody binding epitope in the fVIII C2 domain is highly conserved amongst the two binary complexes determined in this study along with the previously characterized epitope from the C2 domain/3E6/G99 FAB ternary complex28. For each binary complex, all residues proximal to the binding interface are fit within defined electron density (Supplementary Figure S1). The extent of buried surface area between the two binary complexes and the C2/3E6 components of the ternary complex are not significantly different. The C2 domain epitope consists of two loops, Glu2181-Ala2188 and Thr2202-Arg2215 (Fig. 3a,b). Backbone conformations of resdiues proximal to the binding interface do not change significantly, as is the same for the sidechains of His2211, Gln2213, Lys2183, Arg2209 and Asp2187, all of which contribute directly to the 3E6-binding interface. Conformational changes are present, however, for the sidechain of Arg2215 (Fig. 3c). In the ternary structure,.