The crystal structure of IgE Fc reveals an asymmetrically bent conformation

Wan, Tommy; Beavil, Rebecca L.; Fabiane, Stella M.; Beavil, Andrew J.; Sohi, Maninder K.; Keown, Maura B.; Young, Robert J.; Henry, Alistair J.; Owens, Raymond J.; Gould, Hannah J.; Sutton, Brian J.

doi:10.1038/ni811

Cited by 156 publications

(190 citation statements)

References 48 publications

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“…S1 A). However, it is unexpected here since the C 2-C 2 interaction is actually predominantly polar (19), and so the non-polar character in the C 2-C 2 association is not a trivial consequence of the equivalence of the corresponding residues in the contact region. In addition, there were three salt bridges identified within the C 2-C 3,4 interface (19) and (Fig.…”

Section: Resultsmentioning

confidence: 86%

“…However, subsequent fluorescence data of the homologous IgE molecule in solution seemed to indicate that the IgE Fc was actually sharply bent (18). These initial observations were then clearly detailed when the crystallographic structure of the IgE Fc was solved (19), showing that the C 2 domains are bent back toward the C 4 domains by Ϸ60°. These findings raise the possibility that the homologous IgM Fc region may also be bent, although it is not immediately obvious whether such a bent structure could possibly be assembled into a pentameric complex that is planar and in which all of the known disulfide bridges both within and between monomers are satisfied (20).…”

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confidence: 98%

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The human IgM pentamer is a mushroom-shaped molecule with a flexural bias

Czajkowsky

Shao

2009

Proc. Natl. Acad. Sci. U.S.A.

177

146

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The textbook planar model of pentameric IgM, a potent activator of complement C1q, is based upon the crystallographic structure of IgG. Although widely accepted, key predictions of this model have not yet been directly confirmed, which is particularly important since IgG lacks a major Ig fold domain in its Fc region that is present in IgM. Here, we construct a homology-based structural model of the IgM pentamer using the recently obtained crystallographic structure of IgE Fc, which has this additional Ig domain, under the constraint that all of the cysteine residues known to form disulfide bridges both within each monomer and between monomers are bonded together. In contrast to the planar model, this model predicts a non-planar, mushroom-shaped complex, with the central portion formed by the C-terminal domains protruding out of the plane formed by the Fab domains. This unexpected conformation of IgM is, however, directly confirmed by cryo-atomic force microscopy of individual human IgM molecules. Further analysis of this model with free energy calculations of out-of-plane Fab domain rotations reveals a pronounced asymmetry favoring flexions toward the central protrusion. This bias, together with polyvalent attachment to cell surface antigen, would ensure that the IgM pentamer is oriented on the cell membrane with its C1q binding sites fully exposed to the solution, and thus provides a mechanistic explanation for the first steps of C1q activation by IgM.AFM ͉ homology modeling ͉ immunoglobulin ͉ single molecule P entameric IgM is an important component of the first line of defense against foreign pathogens (1, 2) and possibly modified self-components (3), and is increasingly being developed in the diagnosis and therapy of malignancies (4-7). It is also implicated in the damage to organs and tissues following ischemia/reperfusion (8) and in multiple autoimmune diseases (9-11). Its best understood mechanism of action in the immune response is as the initiating component in the classical complement pathway mediated by C1q (12). In this, the binding of IgM to cell surface antigen enables C1q to bind to IgM, which thereby activates C1q for interactions with downstream components. The expected structural changes consequent to antigen-binding and associated with C1q activation, and how these might be modified in disease-conditions, have spurred a longstanding interest in the structure of this large multicomponent molecule (13-17).Like other antibodies, IgM monomers consist of two light and two heavy chains. However, whereas the heavy chains of most antibodies (such as IgG) contain three constant Ig domains, the heavy chains of IgM have a fourth one, as do the heavy chains in IgE. These extra (C 2) domains are located in place of the proline-rich hinge region that is responsible for the rotational flexibility of the antigen-binding Fab domains (relative to the Fc domain).Five IgM monomers complex with an additional small polypeptide (the J chain) to form the predominant oligomeric species in the human plasma (3). Obtaini...

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Section: Resultsmentioning

confidence: 86%

mentioning

confidence: 98%

The human IgM pentamer is a mushroom-shaped molecule with a flexural bias

Czajkowsky

Shao

2009

Proc. Natl. Acad. Sci. U.S.A.

177

146

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“…Flexibility in the Cε3-Cε4 interdomain angle has been welldocumented through studies of unliganded Fcε3-4 structures in different crystal forms (26,27), as well as the unliganded IgE-Fc structure (28) and the sFcεRIα complexes with both Fcε3-4 and IgE-Fc (20,21). Among all of these structures the interdomain angle varies over a range of ∼25°between the most "open" (in the sFcεRIα complexes) and the most "closed" (chain D in the derCD23 complex reported here) (Fig.…”

Section: Resultsmentioning

confidence: 99%

Crystal structure of IgE bound to its B-cell receptor CD23 reveals a mechanism of reciprocal allosteric inhibition with high affinity receptor FcεRI

Dhaliwal

Yuan

Pang

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

136

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The role of IgE in allergic disease mechanisms is performed principally through its interactions with two receptors, FcεRI on mast cells and basophils, and CD23 (FcεRII) on B cells. The former mediates allergic hypersensitivity, the latter regulates IgE levels, and both receptors, also expressed on antigen-presenting cells, contribute to allergen uptake and presentation to the immune system. We have solved the crystal structure of the soluble lectin-like "head" domain of CD23 (derCD23) bound to a subfragment of IgE-Fc consisting of the dimer of Cε3 and Cε4 domains (Fcε3-4). One CD23 head binds to each heavy chain at the interface between the two domains, explaining the known 2:1 stoichiometry and suggesting mechanisms for crosslinking membrane-bound trimeric CD23 by IgE, or membrane IgE by soluble trimeric forms of CD23, both of which may contribute to the regulation of IgE synthesis by B cells. The two symmetrically located binding sites are distant from the single FcεRI binding site, which lies at the opposite ends of the Cε3 domains. Structural comparisons with both free IgE-Fc and its FcεRI complex reveal not only that the conformational changes in IgE-Fc required for CD23 binding are incompatible with FcεRI binding, but also that the converse is true. The two binding sites are allosterically linked. We demonstrate experimentally the reciprocal inhibition of CD23 and FcεRI binding in solution and suggest that the mutual exclusion of receptor binding allows IgE to function independently through its two receptors.antibody-receptor interactions | X-ray crystallography

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“…Finally, we report the crystal structure of the antibody adalimumab Fab with the EFab domain substitution. In comparison with the previously reported adalimumab Fab and IgE Fc structures, 18,19 the adalimumab EFab shows a high degree of similarity in the variable domains. However, clear differences in the relative domain orientation of variable domains with respect to constant domains are observed that do not appear to have a detrimental effect on target antigen binding.…”

Section: Introductionmentioning

confidence: 47%

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

Cooke

Arndt

Quan

et al. 2018

mAbs

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Bispecific antibody therapeutics can expand the functionality of a conventional monoclonal antibody drug because they can bind multiple antigens. However, their great potential is counterbalanced by the challenges faced in their production. The classic asymmetric bispecific containing an Fc requires the expression of four unique chains – two light chains and two heavy chains; each light chain must pair with its correct heavy chain, which then must heterodimerize to form the full bispecific. The light-chain pairing problem has several solutions, some of which require engineering and optimization for each bispecific pair. Here, we introduce a technology called EFab Domain Substitution, which replaces the Cϵ2 of IgE for one of the CL/CH1 domains into one arm of an asymmetric bispecific to encourage the correct pairing of the light chains. EFab Domain Substitution provides very robust correct pairing while maintaining antibody function and is effective for many variable domains. We report its effect on the biophysical properties of an antibody and the crystal structure of the EFab domain substituted into the adalimumab Fab (PDB ID 6CR1).

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The crystal structure of IgE Fc reveals an asymmetrically bent conformation

Cited by 156 publications

References 48 publications

The human IgM pentamer is a mushroom-shaped molecule with a flexural bias

The human IgM pentamer is a mushroom-shaped molecule with a flexural bias

Crystal structure of IgE bound to its B-cell receptor CD23 reveals a mechanism of reciprocal allosteric inhibition with high affinity receptor FcεRI

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

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