An Engineered Disulfide Bond Reversibly Traps the IgE-Fc3–4 in a Closed, Nonreceptor Binding Conformation

Beth A Wurzburg,Beomkyu Kim,Svetlana S Tarchevskaya,Alexander Eggel,Monique Vogel,Theodore S Jardetzky

doi:10.1074/jbc.m112.407502

Beth A Wurzburg, Beomkyu Kim + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m112.407502

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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.

Highlights

IgE antibodies bind the high affinity receptor on mast cells and basophils and trigger allergic diseases
IgE antibodies interact with the high affinity IgE Fc receptor, Fc⑀RI, and activate inflammatory pathways associated with the allergic response
We present the crystal structure of the conformationally trapped IgE-Fc Cys335 protein, and we demonstrate that the Cys-335 Fc does not bind to Fc⑀RI␣ unless the disulfide bond is reduced, freeing the IgE to undergo a conformational change required for receptor binding

Summary

Background

IgE antibodies bind the high affinity receptor on mast cells and basophils and trigger allergic diseases. The structure of the IgE-Fc3–4 in complex with the low affinity IgE receptor (CD23) has been determined, demonstrating that CD23 binds to the C⑀3-4 hinge region, favoring a closed conformation for the Fc [18] These crystallographic observations suggest that conformational dynamics of the IgE affect interactions between IgE and its receptors [9, 13, 16]. These structures indicate that one might be able to regulate IgE conformational dynamics, using protein or small molecules, providing a novel strategy for the development of inhibitors of the IgE-Fc⑀RI interaction [13] To further explore this possibility and to develop new reagents for the identification and isolation of IgE-Fc conformational modulators, we have produced a disulfide bond mutant of the IgE-Fc (Cys-335) that is “trapped” in the closed state. In contrast to its inability to bind to Fc⑀RI, Cys-335 IgE binds two other inhibitory ligands, omalizumab and DARPin E2_79 (19 –21), to wild type IgE, demonstrating a selective block in Fc⑀RI binding

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DISCUSSION