The evolution of flexibility and function in the Fc domains of IgM, IgY, and IgE

Abstract

Introduction: Antibody Fc regions harbour the binding sites for receptors that mediate effector functions following antigen engagement by the Fab regions. An extended "hinge" region in IgG allows flexibility between Fab and Fc, but in both the most primitive antibody, IgM, and in the evolutionarily more recent IgE, the hinge is replaced by an additional domain pair in the homodimeric six-domain Fc region. This permits additional flexibility within the Fc region, which has been exploited by nature to modulate antibody effector functions. Thus, in pentameric or hexameric IgM, the Fc regions appear to adopt a planar conformation in solution until antigen binding causes a conformational change and exposes the complement binding sites. In contrast, IgE-Fc principally adopts an acutely bent conformation in solution, but the binding of different receptors is controlled by the degree of bending, and there is allosteric communication between receptor binding sites.

Methods: We sought to trace the evolution of Fc conformational diversity from IgM to IgE via the intermediate avian IgY by studying the solution conformations of their Fc regions by small-angle X-ray scattering. We compared four extant proteins: human IgM-Fc homodimer, chicken IgY-Fc, platypus IgE-Fc, and human IgE-Fc. These are examples of proteins that first appeared in the jawed fish [425 million years ago (mya)], tetrapod (310 mya), monotreme (166 mya), and hominid (2.5 mya) clades, respectively.

Results and discussion: We analysed the scattering curves in terms of contributions from a pool of variously bent models chosen by a non-negative linear least-squares algorithm and found that the four proteins form a series in which the proportion of acutely bent material increases: IgM-Fc < IgY-Fc < plIgE-Fc < huIgE-Fc. This follows their order of appearance in evolution. For the huIgM-Fc homodimer, although none are acutely bent, and a significant fraction of the protein is sufficiently bent to expose the C1q-binding site, it predominantly adopts a fully extended conformation. In contrast, huIgE-Fc is found principally to be acutely bent, as expected from earlier studies. IgY-Fc, in this first structural analysis of the complete Fc region, exhibits an ensemble of conformations from acutely bent to fully extended, reflecting IgY's position as an evolutionary intermediate between IgM and IgE.

Keywords: Fc region; IgM; IgY; evolution; flexibility; human IgE; platypus IgE; receptor.

Figure 1

(A) Schematic structure and domain nomenclature for the heavy and light chains of an antibody such as IgY or IgE, which consists of six domains in the Fc region. The antigen-binding Fab region is also shown. (B) Schematic of the domain structure and nomenclature of pentameric IgM with the J-chain, in the absence of which, a hexamer of the H₂L₂ subunits forms. The IgM-Fc region of a single subunit, such as that indicated by the domain nomenclature, consisting of six domains, enables direct comparison with IgY-Fc and IgE-Fc. The complement binding sites in IgM lie in the Cμ3 domains close to the connection with the Cμ2 domains; they are not indicated here but are illustrated schematically in Figure 2 . Disulphide bridges within and between subunits, and the tail-piece extensions of the H chains that engage with the J-chain, are not shown.

Figure 2

(A) Schematic structure of a part of two subunits of IgM (which themselves are part of a pentamer), bent between their Cμ2 and Cμ3 domains to allow two Fab regions to engage with a multi-valent antigen (bamboo colour). The figure is adapted from cryo-electron tomography structures presented in ref. (7). Only one Fab from each IgM subunit is shown (pale pink; the other is omitted for clarity), and the Fc regions (pink) of the two subunits are shown in contact through their Cμ4 domains (as shown schematically for the pentamer in Figure 1B ). The bending of the Fc regions at the Cμ3-Cμ2 interface exposes binding sites for C1q head domains in the Cμ3 domains as indicated, and the overall bend of the Fab arms relative to the plane of the Fc₅ region is 100°C. (B) Schematic illustrations of the domains of IgE-Fc when bound to (i) FcεRIα and (ii) CD23 (PDB 2Y7Q and PDB 4EZM, respectively). In both, there is an acute bend between the Cε2 and Cε3 domains, which is slightly more pronounced in the former and accompanied by an “opening” of the Cε3 domains. (C) Schematic illustration of the domain structure of IgY-Fc with the receptors i) ggFcR and ii) CHIR-AB1 bound. Note that the disposition of the Cυ2 domains is unknown in free or receptor-bound IgY-Fc, and the locations of the receptor sites are only implied by homology and mutagenesis evidence (refer to the text). The extent of any flexibility (indicated by the double-headed arrows) of the Cυ2 domains relative to the rest of the Fc is also unknown, prior to the present study.

Figure 3

Comparison of SAXS analyses of the four recombinant Fc proteins (Preparation 1) using Scåtter. (A) Intensity plot. (B) Guinier plot. (C) P(r) plot. (D) Dimensionless Kratky plot. In panels (A, B) the first point on each plot has been transposed to (0,0) to facilitate a comparison of the Rg values for the four proteins huIgM-Fc (pink), chIgY-Fc (blue), plIgE-Fc (green), and huIgE-Fc (yellow).

Figure 4

Models selected to best fit the X-ray scattering data for the four recombinant Fc proteins (Preparation 1). Relative contributions and binned Rg values for each of the models selected for human huIgM-Fc (pink), chIgY-Fc (blue), plIgE-Fc (green), and huIgE-Fc (yellow) are shown. Four bins were chosen to cover the range of Rg values from acutely bent (28-31Å), to partially bent (31-34Å and 34-37Å) to fully extended (37-40Å). The number of models contributing to each bar is indicated.