title: An engineered disulfide bond reversibly traps the IgE-Fc3-4 in a closed, nonreceptor binding conformation.
abstract: IgE antibodies interact with the high affinity IgE Fc receptor, FcεRI, and activate inflammatory pathways associated with the allergic response. The IgE-Fc region, comprising the C-terminal domains of the IgE heavy chain, binds FcεRI and can adopt different conformations ranging from a closed form incompatible with receptor binding to an open, receptor-bound state. A number of intermediate states are also observed in different IgE-Fc crystal forms. To further explore this apparent IgE-Fc conformational flexibility and to potentially trap a closed, inactive state, we generated a series of disulfide bond mutants. Here we describe the structure and biochemical properties of an IgE-Fc mutant that is trapped in the closed, non-receptor binding state via an engineered disulfide at residue 335 (Cys-335). Reduction of the disulfide at Cys-335 restores the ability of IgE-Fc to bind to its high affinity receptor, FcεRIα. The structure of the Cys-335 mutant shows that its conformation is within the range of previously observed, closed form IgE-Fc structures and that it retains the hydrophobic pocket found in the hinge region of the closed conformation. Locking the IgE-Fc into the closed state with the Cys-335 mutation does not affect binding of two other IgE-Fc ligands, omalizumab and DARPin E2_79, demonstrating selective blocking of the high affinity receptor binding.
authors.affiliation: Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94305, USA.
authors.forename: Beth A
authors.initials: BA
authors.lastname: Wurzburg
authors.forename: Beomkyu
authors.initials: B
authors.lastname: Kim
authors.forename: Svetlana S
authors.initials: SS
authors.lastname: Tarchevskaya
authors.forename: Alexander
authors.initials: A
authors.lastname: Eggel
authors.forename: Monique
authors.initials: M
authors.lastname: Vogel
authors.forename: Theodore S
authors.initials: TS
authors.lastname: Jardetzky
mesh_terms: D019943:Amino Acid Substitution; D000888:Antibodies, Anti-Idiotypic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D000069444:Omalizumab; D011485:Protein Binding / Q000235:genetics; D020836:Protein Structure, Quaternary; D017455:Receptors, IgE / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D000888:Antibodies, Anti-Idiotypic; D061067:Antibodies, Monoclonal, Humanized; D004220:Disulfides; D007143:Immunoglobulin Heavy Chains; D017455:Receptors, IgE; D000069444:Omalizumab