title: A fluorescent biosensor reveals conformational changes in human immunoglobulin E Fc: implications for mechanisms of receptor binding, inhibition, and allergen recognition.
abstract: IgE binding to its high affinity receptor FcεRI on mast cells and basophils is a key step in the mechanism of allergic disease and a target for therapeutic intervention. Early indications that IgE adopts a bent structure in solution have been confirmed by recent x-ray crystallographic studies of IgEFc, which further showed that the bend, contrary to expectation, is enhanced in the crystal structure of the complex with receptor. To investigate the structure of IgEFc and its conformational changes that accompany receptor binding in solution, we created a Förster resonance energy transfer (FRET) biosensor using biologically encoded fluorescent proteins fused to the N- and C-terminal IgEFc domains (Cε2 and Cε4, respectively) together with the theoretical basis for quantitating its behavior. This revealed not only that the IgEFc exists in a bent conformation in solution but also that the bend is indeed enhanced upon FcεRI binding. No change in the degree of bending was seen upon binding to the B cell receptor for IgE, CD23 (FcεRII), but in contrast, binding of the anti-IgE therapeutic antibody omalizumab decreases the extent of the bend, implying a conformational change that opposes FcεRI engagement. HomoFRET measurements further revealed that the (Cε2)(2) and (Cε4)(2) domain pairs behave as rigid units flanking the conformational change in the Cε3 domains. Finally, modeling of the accessible conformations of the two Fab arms in FcεRI-bound IgE revealed a mutual exclusion not seen in IgG and Fab orientations relative to the membrane that may predispose receptor-bound IgE to cross-linking by allergens.
authors.affiliation: MRC and Asthma UK Centre in Allergic Mechanisms of Asthma, Guy's Hospital Campus, London SE1 1UL; The Randall Division of Cell and Molecular Biophysics, Guy's Hospital Campus, London SE1 1UL; The Division of Asthma Allergy and Lung Biology, King's College London, Guy's Hospital Campus, London SE1 1UL.
authors.forename: James
authors.initials: J
authors.lastname: Hunt
authors.affiliation: MRC and Asthma UK Centre in Allergic Mechanisms of Asthma, Guy's Hospital Campus, London SE1 1UL; The Randall Division of Cell and Molecular Biophysics, Guy's Hospital Campus, London SE1 1UL; The Division of Asthma Allergy and Lung Biology, King's College London, Guy's Hospital Campus, London SE1 1UL.
authors.forename: Anthony H
authors.initials: AH
authors.lastname: Keeble
authors.affiliation: The Randall Division of Cell and Molecular Biophysics, Guy's Hospital Campus, London SE1 1UL.
authors.forename: Robert E
authors.initials: RE
authors.lastname: Dale
authors.affiliation: The Randall Division of Cell and Molecular Biophysics, Guy's Hospital Campus, London SE1 1UL.
authors.forename: Melissa K
authors.initials: MK
authors.lastname: Corbett
authors.affiliation: MRC and Asthma UK Centre in Allergic Mechanisms of Asthma, Guy's Hospital Campus, London SE1 1UL; The Randall Division of Cell and Molecular Biophysics, Guy's Hospital Campus, London SE1 1UL; The Division of Asthma Allergy and Lung Biology, King's College London, Guy's Hospital Campus, London SE1 1UL.
authors.forename: Rebecca L
authors.initials: RL
authors.lastname: Beavil
authors.affiliation: The Department of Physics, King's College London, Strand, London WC2R 2LS.
authors.forename: James
authors.initials: J
authors.lastname: Levitt
authors.affiliation: Farfield Group Limited, Voyager, Chicago Avenue, Manchester Airport, Manchester, M90 3DQ, United Kingdom.
authors.forename: Marcus J
authors.initials: MJ
authors.lastname: Swann
authors.affiliation: The Department of Physics, King's College London, Strand, London WC2R 2LS.
authors.forename: Klaus
authors.initials: K
authors.lastname: Suhling
authors.affiliation: The Randall Division of Cell and Molecular Biophysics, Guy's Hospital Campus, London SE1 1UL.
authors.forename: Simon
authors.initials: S
authors.lastname: Ameer-Beg
authors.affiliation: MRC and Asthma UK Centre in Allergic Mechanisms of Asthma, Guy's Hospital Campus, London SE1 1UL; The Randall Division of Cell and Molecular Biophysics, Guy's Hospital Campus, London SE1 1UL.
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Sutton
authors.affiliation: MRC and Asthma UK Centre in Allergic Mechanisms of Asthma, Guy's Hospital Campus, London SE1 1UL; The Randall Division of Cell and Molecular Biophysics, Guy's Hospital Campus, London SE1 1UL; The Division of Asthma Allergy and Lung Biology, King's College London, Guy's Hospital Campus, London SE1 1UL. Electronic address: andrew.beavil@kcl.ac.uk.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Beavil
mesh_terms: D000485:Allergens / Q000032:analysis*; D000888:Antibodies, Anti-Idiotypic / Q000737:chemistry; D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry; D015374:Biosensing Techniques / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D007073:Immunoglobulin E / Q000737:chemistry* / Q000235:genetics; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000235:genetics; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry*; D000069444:Omalizumab; D017455:Receptors, IgE / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D000485:Allergens; D000888:Antibodies, Anti-Idiotypic; D061067:Antibodies, Monoclonal, Humanized; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D017455:Receptors, IgE; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000069444:Omalizumab; D007073:Immunoglobulin E