Thermodynamics of the Interaction of Human Immunoglobulin E with Its High-Affinity Receptor FcεRI

Abstract

We have employed isothermal titration calorimetry (ITC) and circular dichroism (CD) spectroscopy to characterize the binding of soluble fragments of IgE (IgE-Fc and Fc epsilon 3-4) to a soluble fragment of the high-affinity receptor Fc epsilon RI alpha-chain (sFc epsilon RI alpha). The thermodynamic parameters for the interaction of IgE-Fc and Fc epsilon 3-4 with sFc epsilon RI alpha, determined using ITC, confirm the earlier conclusion that the C epsilon 2 domain is not involved in the interaction and that the stoichiometry of both complexes is 1:1. For both IgE-Fc and Fc epsilon 3-4, the value of Delta H degrees is -36.9 +/- 4.6 kcal mol-1 at 37.3 degreesC and Delta Cp degrees is -820 +/- 120 cal mol-1 K-1. The temperature at which DeltaS degrees is zero is 284 +/- 1 K, indicating that the entropy contribution to the thermodynamics of association is unfavorable at physiological temperature. Of particular interest is the large value of Delta Cp degrees. The large surface area of IgE and Fc epsilon RI alpha that is implicated in complex formation from previous mutagenesis studies on the two proteins may account in part for the magnitude of Delta Cp degrees. Additional contributions may arise from hydration within the binding site and changes in tertiary structure of the individual components of the complex. However, the CD spectra of IgE, IgE-Fc, and Fc epsilon 3-4 complexes with sFc epsilon RI alpha are merely the sum of the spectra of their individual components, indicating that the secondary structure of the immunoglobulin domain folds are preserved on complex formation. Thus, any change in tertiary structure must be limited to the relative disposition of the immunoglobulin domains C epsilon 3 and C epsilon 4 in IgE and the two immunoglobulin-like domains in the alpha-chain of Fc epsilon RI.

Disulfide Linkage Controls the Affinity and Stoichiometry of IgE Fcϵ3–4 Binding to FcϵRI

“…3) were all typical of a folded protein consisting principally of ␤-structure and in agreement with data previously published for Fc⑀3-4 fragments (10,14,20). This indicates that neither the refolding of the protein expressed in E. coli nor deglycosylation of the NS-0 material affected the folding of the fragment.…”

“…We have analyzed these effects using four variants of the Fc⑀3-4 fragment: (i) disulfide-linked and glycosylated (Fc⑀3-4); (ii) lacking only the disulfide link (redFc⑀3-4); (iii) lacking only glycosylation (deglyFc⑀3-4); and (iv) lacking both structural features (Fc⑀3-4⌬C). Fc⑀3-4 lacks the C⑀2 domains of the complete IgE Fc, but we have shown previously that not only does this fragment display a 1:1 stoichiometry of binding (9), it also retains full binding affinity (10), although the kinetics of binding are affected (3). It has been reported previously that deglycosylation of IgE has little effect upon receptor binding (11) and that E. coli expressed fragments retain high-affinity receptor binding activity (12).…”

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

“…The ⌬C P 0 (Ϫ760 cal mol Ϫ1 K Ϫ1 ) and ⌬H A 0 (25°C, Ϫ20.4 kcal mol and found to be similar to previously determined values (35). The remaining thermodynamic parameters derived by DSC for determination of the apparent binding affinity of Fc⑀ to Fc␥-Fc⑀RI␣ are shown in Table 1.…”

Interaction of Human IgE with Fc Epsilon RI Alpha Exposes Hidden Epitopes on IgE

“…Experimental evidence supporting the former has been provided by circular dichroism spectroscopy [9]whereas indications for the latter exist from neutron–scattering data [7]. Recently, Keown et al [23]reported that no change in secondary structure occurs upon IgE–FcεRI complex formation and therefore postulated that tertiary structure changes must be limited to the relative disposition of Cε3 and Cε4 of IgE–Fc or the immunoglobulin–like domains of FcεRIα.…”