title: Characterization and solution structure of the factor VIII C2 domain in a ternary complex with classical and non-classical inhibitor antibodies.
abstract: The most significant complication for patients with severe cases of congenital or acquired hemophilia A is the development of inhibitor antibodies against coagulation factor VIII (fVIII). The C2 domain of fVIII is a significant antigenic target of anti-fVIII antibodies. Here, we have utilized small angle x-ray scattering (SAXS) and biochemical techniques to characterize interactions between two different classes of anti-C2 domain inhibitor antibodies and the isolated C2 domain. Multiple assays indicated that antibodies 3E6 and G99 bind independently to the fVIII C2 domain and can form a stable ternary complex. SAXS-derived numerical estimates of dimensional parameters for all studied complexes agree with the proportions of the constituent proteins. Ab initio modeling of the SAXS data results in a long kinked structure of the ternary complex, showing an angle centered at the C2 domain of ∼130°. Guided by biochemical data, rigid body modeling of subunits into the molecular envelope of the ternary complex suggests that antibody 3E6 recognizes a C2 domain epitope consisting of the Arg(2209)-Ser(2216) and Leu(2178)-Asp(2187) loops. In contrast, antibody G99 recognizes the C2 domain primarily through the Pro(2221)-Trp(2229) loop. These two epitopes are on opposing sides of the fVIII C2 domain, are consistent with the solvent accessibility in the context of the entire fVIII molecule, and provide further structural detail regarding the pathogenic immune response to fVIII.
authors: Department of Chemistry, Western Washington University, Bellingham, Washington 98225.
authors: Justin D
authors: JD
authors: Walter
authors: Department of Chemistry, Western Washington University, Bellingham, Washington 98225.
authors: Rachel A
authors: RA
authors: Werther
authors: Department of Chemistry, Western Washington University, Bellingham, Washington 98225.
authors: Maria S
authors: MS
authors: Polozova
authors: Department of Chemistry, Western Washington University, Bellingham, Washington 98225.
authors: Julie
authors: J
authors: Pohlman
authors: Aflac Cancer and Blood Disorders Center, Department of Pediatrics, Emory University, Atlanta, Georgia 30322.
authors: John F
authors: JF
authors: Healey
authors: Aflac Cancer and Blood Disorders Center, Department of Pediatrics, Emory University, Atlanta, Georgia 30322.
authors: Shannon L
authors: SL
authors: Meeks
authors: Aflac Cancer and Blood Disorders Center, Department of Pediatrics, Emory University, Atlanta, Georgia 30322.
authors: Pete
authors: P
authors: Lollar
authors: Department of Chemistry, Western Washington University, Bellingham, Washington 98225. Electronic address: spiegep@chem.wwu.edu.
authors: P Clint
authors: PC
authors: Spiegel
mesh_terms: D001779:Blood Coagulation Factors / Q000737:chemistry; D002845:Chromatography / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D000939:Epitopes / Q000737:chemistry; D005169:Factor VIII / Q000737:chemistry* / Q000378:metabolism; D006467:Hemophilia A / Q000276:immunology; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D053838:Scattering, Small Angle; D012997:Solvents / Q000737:chemistry
chemical_list: D001779:Blood Coagulation Factors; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D012997:Solvents; D005169:Factor VIII