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

Abstract

Of all the antibody classes, IgE displays a uniquely slow dissociation rate from, and high affinity for, its cell surface receptor FcεRI. The structural basis for these key determinants of IgE's ability to mediate allergic hypersensitivity is now revealed by the 3.4Å resolution crystal structure of human IgE-Fc (consisting of the Cε2, Cε3 and Cε4 domains) bound to the extracellular domains of the FcεRI α-chain. Comparison with free IgE-Fc (reported here at 1.9Å) shows that the antibody, which has a compact, bent structure prior to receptor engagement, becomes even more acutely bent in the complex. Thermodynamic analysis indicates that the interaction is entropically driven, which explains how the non-contacting Cε2 domains, in place of the flexible hinge region of IgG antibodies, contribute together with the conformational changes to IgE's unique binding properties.The global incidence of allergic disease has increased markedly in recent years and continues to rise. Asthma currently affects 22.2 million people in North America and 5.4 million in the UK where the rate of incidence, especially in children, is among the highest in the world 1 . Anaphylactic reactions to foods such as nuts, virtually unknown thirty years ago, are now relatively common. The reason for this increase is debated, but these and other allergic conditions (rhinitis, eczema etc.) are all mediated by IgE, and the viability of AUTHOR CONTRIBUTIONS MDH and AMD carried out the crystallographic analysis of the complex, and BD the crystallographic analysis of IgE-Fc; MDH, JEN, JH, AJB and RJO produced the proteins; SCB and JMM carried out the thermodynamic analysis; EG contributed to the analysis of the conformational changes; HJG, AJB and BJS planned and directed the project; MDH, AMD, JMM and BJS wrote the paper.Accession codes. Atomic coordinates and structure factors have been deposited in the Protein Data Bank with entry codes 2WQR for IgE-Fc and 2Y7Q for the complex. [6][7][8][9] . Particular interest has centered on the role of the Cε2 domains, which were shown to contribute to the slow dissociation rate 10 , by comparing the binding kinetics of the complete IgE-Fc (dimer Cε2-Cε3-Cε4 domains) with a sub-fragment lacking the Cε2 domains, here referred to as Fcε3-4. The Cε2 domains have no counterpart in IgG, all four subclasses of which have in their place hinge regions of various lengths and degrees of flexibility. Europe PMC Funders GroupEarly fluorescence resonance energy transfer (FRET) studies of labelled chimeric IgE indicated a more compact and bent structure than the extended, flexible Y-shaped IgG 11 , and later solution scattering studies of IgE-Fc were consistent with such a structure 12 . The crystal structure of IgE-Fc revealed for the first time the extent and nature of this bend 13 , made possible by the presence of the Cε2 domains. Surprisingly the molecule was found to be so acutely and asymmetrically bent, with the Cε2 domain pair folded back across the Cε3 domains, that one Cε2 domain even contacted the Cε4 domai...

EFab domain substitution as a solution to the light-chain pairing problem of bispecific antibodies

“…19 A superposition of the VH and VL domains of EFab and Fab show a root mean square deviation (rmsd) of 0.37 Å (Cα atoms only) (Figure 5B), demonstrating the high structural similarity. In contrast, when the Cϵ2 domains of the EFab are superimposed to the Cϵ2 domains of the IgE Fc (PDB id: 2WQR) 16 the rmsd of the Cα atoms is 1.32 Å, indicating less structural similarity (Figure 5B). …”

“…(B) Top, superposition of variable domains in EFab and Fab with a C-alpha rmsd of 0.37 Å. Bottom, superposition of the Cϵ2 domains in EFab and IgE Fc (green, PDB ID: 2WQR) 16 with an rmsd of 1.32 Å. (C) Relative domain orientation of variable and constant domains in EFab and Fab.…”

“…It is worthy of note that the Cϵ2 dimer is well defined in the IgE Fc structure. 16 It is unlikely that this disorder is caused by the knob-and-hole heterodimerization mutations in the EFab design because the Cϵ2 heterodimer interface shows clear electron density in an unbiased composite omit maps (Figure 6), indicating a defined geometry that matches well with the WT Fab. The disordered region is more likely to be explained by the structural constraints imposed by the Cϵ2 domains being fused to the variable region.

10.1080/19420862.2018.1519631-F0006Figure 6.Composite omit map around the knob-and-hole mutations at the Cϵ2 – Cϵ2 interface of the EFab, contoured at 1.2 sigma using a carve distance of 1.6 Å.

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“…30 Separate search models were the Fab variable domains of adalimumab Fab (PDB id 4NYL) and IgE Cϵ2 domains from an IgE Fc (PDB id 2WQR). 16 The adalimumab EFab model was rebuilt using with Coot and refinement using REFMAC resulted in placement of nearly all the adalimumab EFab residues except for the His tag. The final stages of refinement employed TLS refinement with anisotropic motion tensors refined for each of the domains, using REFMAC.…”

“…Thus, unlike the CH2 domain of IgG, the Cɛ2 domain is not involved in effector function and makes no contact with the FcɛRIα. 16 Interestingly, the homodimers of IgM or IgE CH2 domains have been used as homodimerization scaffolds for fusion proteins. 17 However, the use of Cɛ2 as a heterodimerization platform has not been reported.…”