An Intermediate pH Unfolding Transition Abrogates the Ability of IgE to Interact with Its High Affinity Receptor FcϵRIα

Abstract The interaction between IgE-Fc (Fcϵ) and its high affinity receptor FcϵRI on the surface of mast cells and basophils is a key event in allergen-induced allergic inflammation. Recently, several therapeutic strategies have been developed based on this interaction, and some include Fcϵ-containing moieties. Unlike well characterized IgG therapeutics, the stability and folding properties of IgE are not well understood. Here, we present comparative biophysical analyses of the pH stability and thermostability of Fcϵ and IgG1-Fc (Fcγ). Fcϵ was found to be significantly less stable than Fcγ under all pH and NaCl conditions tested. Additionally, the Cϵ3Cϵ4 domains of Fcϵ were shown to become intrinsically unfolded at pH values below 5.0. The interaction between Fcϵ and an Fcγ-FcϵRIα fusion protein was studied between pH 4.5 and 7.4 using circular dichroism and a combination of differential scanning calorimetry and isothermal titration calorimetry. Under neutral pH conditions, the apparent affinity of Fcϵ for the dimeric fusion protein was extremely high compared with published values for the monomeric receptor (KD < 10-12 m). Titration to pH 6.0 did not significantly change the binding affinity, and titration to pH 5.5 only modestly attenuated affinity. At pH values below 5.0, the receptor binding domains of Fcϵ unfolded, and interaction of Fcϵ with the Fcγ-FcϵRIα fusion protein was abrogated. The unusual pH sensitivity of Fcϵ may play a role in antigen-dependent regulation of receptor-bound, non-circulating IgE.