Conformational changes in IgE contribute to its uniquely slow dissociation rate from receptor FcɛRI

Abstract

Among antibody classes, IgE has a uniquely slow dissociation rate from, and high affinity for, its cell surface receptor FcɛRI. We show the structural basis for these key determinants of the ability of IgE to mediate allergic hypersensitivity through the 3.4-Å-resolution crystal structure of human IgE-Fc (consisting of the Cɛ2, Cɛ3 and Cɛ4 domains) bound to the extracellular domains of the FcɛRI α chain. Comparison with the structure of free IgE-Fc (reported here at a resolution of 1.9 Å) shows that the antibody, which has a compact, bent structure before receptor engagement, becomes even more acutely bent in the complex. Thermodynamic analysis indicates that the interaction is entropically driven, which explains how the noncontacting Cɛ2 domains, in place of the flexible hinge region of IgG antibodies, contribute together with the conformational changes to the unique binding properties of IgE.

Figure 1

Overall structure of the IgE-Fc/sFcεRIα complex, in two approximately orthogonal views. sFcεRIα is colored in blue, IgE-Fc chain A in yellow and chain B in purple. a) The extensive interface with two distinct sub-sites, one on each Cε3 domain, is indicated with a space-filled representation of the interacting side-chains. The Cε4 domains are hidden in this orientation. b) The acute bend, with the pair of Cε2 domains packed against the Cε3 and Cε4 domains, can be seen clearly. The connections to the Fab regions are indicated, and the bend in the IgE molecule ensures that they are oriented away from the membrane (see also Supplementary Video 1).

Figure 2

Closure of an inter-domain cleft in IgE-Fc upon receptor binding. a) In the free IgE-Fc structure there is a cleft between the first N-acetylglucosamine carbohydrate unit (blue), N-linked to Asn394 (behind) in Cε3B (yellow), and residues Asp271 and Asp307 in Cε2B (orange). b) In the complex, the movement of Cε3A (green), Cε2B (orange) and Cε2A (not shown) as a rigid unit relative to Cε3B (yellow) and the Cε4 domains (not shown) closes the cleft, and Asp271 and carbohydrate make contact, with the formation of two potential H-bonds (black lines). Both figures are centered on the first N-acetylglucosamine unit. For clarity, other carbohydrate residues are not shown for the unbound form (panel a); all carbohydrate units except for the first N-acetylglucosamine were disordered in the complex (panel b).

Figure 3

Interactions at the two sub-sites. a) Two salt bridges (Arg334–Glu132; Asp362–Lys117) with three hydrogen bonds (black lines) between residues of the Cε3A domain of IgE-Fc (yellow) and the receptor (blue) contribute to sub-site 1. b) The “proline sandwich” at sub-site 2, with Pro426 in the Cε3B domain of IgE-Fc (purple) packed between Trp87 and Trp110 of the receptor (blue). The alternative orientation of Trp87 observed in the Fcε3-4/sFcεRIα complex (light grey) can be seen to make fewer contacts and a weaker interaction with Pro426.

Figure 4

Thermodynamics of the IgE/FcεRI interaction. SPR sensorgrams of Fcε2-4 (IgE-Fc) and Fcε3-4 binding to immobilized sFcεRIα wildtype (a & b), and to immobilized sFcεRIα W87D (c & d), over a range of temperatures. A series of analyte concentrations were tested; sensorgrams for a single concentration point (125 nM) are shown for each temperature (see Supplementary Figs. 5 and 6 for full range of concentration data). (e) The van’t Hoff plot illustrates the temperature dependence of the equilibrium binding affinities for Fcε2-4/sFcεRIα wildtype, Fcε2-4/sFcεRIα W87D, Fcε3-4/sFcεRIα wildtype, and Fcε3-4/sFcεRIα W87D. The fits for both Fcε2-4 interactions are linear (R > 0.99), whereas the Fcε3-4 interactions show small deviations from linearity (R = 0.96–0.98), consistent with a minor contribution from ΔCp. The derived thermodynamic parameters are summarized in Table 2.