A Fluorescent Biosensor Reveals Conformational Changes in Human Immunoglobulin E Fc: IMPLICATIONS FOR MECHANISMS OF RECEPTOR BINDING, INHIBITION, AND ALLERGEN RECOGNITION

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Abstract

IgE binding to its high affinity receptor Fc epsilon RI 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 Forster resonance energy transfer (FRET) biosensor using biologically encoded fluorescent proteins fused to the N- and C-terminal IgEFc domains (C epsilon 2 and C epsilon 4, 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 Fc epsilon RI binding. No change in the degree of bending was seen upon binding to the B cell receptor for IgE, CD23 (Fc epsilon RII), but in contrast, binding of the anti-IgE therapeutic antibody omalizumab decreases the extent of the bend, implying a conformational change that opposes Fc epsilon RI engagement. HomoFRET measurements further revealed that the (C epsilon 2)(2) and (C epsilon 4)(2) domain pairs behave as rigid units flanking the conformational change in the C epsilon 3 domains. Finally, modeling of the accessible conformations of the two Fab arms in Fc epsilon RI-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.

Original languageEnglish
Pages (from-to)17459-17470
Number of pages12
JournalJournal of Biological Chemistry
Volume287
Issue number21
Early online date22 Mar 2012
DOIs
Publication statusPublished - 18 May 2012

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