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    Beomkyu Kim.
    IgE antibodies interact with the high affinity IgE Fc receptor (Fc [epsilon] RI) expressed on mast cells and basophils, and the low affinity IgE Fc receptor (CD23) on B cells. These interactions are directly involved in triggering the allergen-specific activation of inflammatory responses. The IgE-Fc region, comprising the C-terminal domains of the IgE heavy chain, binds Fc [epsilon] RI and the crystal structures of the human IgE-Fc alone and IgE-Fc complexes exhibited IgE-Fc conformational flexibility. In addition, the crystal structure of IgE-Fc bound to CD23 indicated a preference of CD23 for a closed conformation of the IgE-Fc incompatible with Fc [epsilon] RI binding. These observations suggested the importance of IgE conformational dynamics for its receptor binding mechanism and the potential to identify non-classical or allosteric inhibitors for IgE-receptor interactions. To investigate IgE dynamics and its role in receptor binding and inhibition, I have employed 5 experimental approaches: 1) IgE-Fc mutants trapped by an engineered disulfide bond, blocking Fc [epsilon] RI but not IgE inhibitor binding, 2) a fluorescence assay for IgE-Fc:Fc [epsilon] RI binding and inhibition that can distinguish IgE-inhibitors based on their proximity to the Fc [epsilon] RI binding site, 3) the determination of the E2_79 crystal structure showing that the IgE inhibitor E2_79 binds IgE-Fc outside the Fc [epsilon] RI-binding site and acts through a facilitated dissociation mechanism to block receptor binding, 4) the development of a TR-FRET assay suitable for high-throughput screening for small molecule inhibitors of IgE-receptor binding, and 5) a single-molecule FRET approach to characterize the conformational states of the IgE-Fc3-4 and observe changes upon Fc [epsilon] RI, CD23, and E2_79. These experiments revealed different conformational requirements for IgE binding, identified a disruptive inhibitor that actively dissociates preformed receptor complexes, and provided direct observation of receptor binding-induced IgE-Fc conformational changes. This work provides a new perspective on the role of IgE dynamics in receptor binding and inhibition, and makes possible the development of new disruptive inhibitors for IgE-receptor binding acting through non-classical or allosteric mechanisms.
    Digital Access   2013