I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[1220, 7656, 9495]
Open Access
Abstract: Immunoglobulin E (IgE) plays a central role in the allergic response, in which cross-linking of allergen by Fc∊RI-bound IgE triggers mast cell and basophil degranulation and the release of inflammatory mediators. The high-affinity interaction between IgE and Fc∊RI is a long-standing target for therapeutic intervention in allergic disease. Omalizumab is a clinically approved anti-IgE monoclonal antibody that binds to free IgE, also with high affinity, preventing its interaction with Fc∊RI. All attempts to crystallize the pre-formed complex between the omalizumab Fab and the Fc region of IgE (IgE-Fc), to understand the structural basis for its mechanism of action, surprisingly failed. Instead, the Fab alone selectively crystallized in different crystal forms, but their structures revealed intermolecular Fab/Fab interactions that were clearly strong enough to disrupt the Fab/IgE-Fc complexes. Some of these interactions were common to other Fab crystal structures. Mutations were therefore designed to disrupt two recurring packing interactions observed in the omalizumab Fab crystal structures without interfering with the ability of the omalizumab Fab to recognize IgE-Fc; this led to the successful crystallization and subsequent structure determination of the Fab/IgE-Fc complex. The mutagenesis strategy adopted to achieve this result is applicable to other intractable Fab/antigen complexes or systems in which Fabs are used as crystallization chaperones.
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Mar 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Jiun-Bo
Chen
,
Faruk
Ramadani
,
Marie O. Y.
Pang
,
Rebecca L.
Beavil
,
Mary D.
Holdom
,
Alkistis N.
Mitropoulou
,
Andrew J.
Beavil
,
Hannah J.
Gould
,
Tse Wen
Chang
,
Brian
Sutton
,
James M.
Mcdonnell
,
Anna M.
Davies
Diamond Proposal Number(s):
[7656]
Open Access
Abstract: Immunoglobulin E (IgE) antibodies play a central role in the allergic response: interaction with FcεRI on mast cells and basophils leads to immediate hypersensitivity reactions upon allergen challenge, while interaction with CD23/FcεRII, expressed on a variety of cells, regulates IgE synthesis among other activities. The receptor-binding IgE-Fc region has recently been found to display remarkable flexibility, from acutely bent to extended conformations, with allosteric communication between the distant FcεRI and CD23 binding sites. We report the structure of an anti-IgE antibody Fab (8D6) bound to IgE-Fc through a mixed protein-carbohydrate epitope, revealing further flexibility and a novel extended conformation with potential relevance to that of membrane-bound IgE in the B cell receptor for antigen. Unlike the earlier, clinically approved anti-IgE antibody omalizumab, 8D6 inhibits binding to FcεRI but not CD23; the structure reveals how this discrimination is achieved through both orthosteric and allosteric mechanisms, supporting therapeutic strategies that retain the benefits of CD23 binding.
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Aug 2018
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9495, 7656]
Abstract: In allergic disease, mast cell activation is conventionally triggered by allergen-mediated cross-linking of receptor-bound IgE on the cell surface. In addition to its diverse range of intracellular roles in apoptosis, cell proliferation and cancer, Histamine-Releasing Factor (HRF) also activates mast cells and basophils. A subset of IgE antibodies bind HRF through their Fab regions, and two IgE binding sites on HRF have been mapped. HRF can form dimers, and a disulphide-linked dimer is critical for activity. The current model for the activity of HRF in mast cell activation involves cross-linking of receptor-bound IgE by dimeric HRF, mediated by HRF/Fab interactions. HRF crystal and solution structures have provided little insight into either the formation of disulphide-linked HRF dimers or the ability of HRF to activate mast cells. We report the first crystal structure of murine HRF (mHRF) to 4.0 Å resolution, revealing a conserved fold. We also solved the structure of human HRF (hHRF) in two new crystal forms, one at the highest resolution (1.4 Å) yet reported. The high resolution hHRF structure reveals a disulphide-linked dimer, in which the two molecules are closely associated, and provides a model for the role of both human and murine HRF in mast cell activation.
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Dec 2017
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[1220, 9495]
Open Access
Abstract: Immunoglobulin E (IgE) is the antibody that plays a central role in the mechanisms of allergic diseases such as asthma. Interactions with its receptors, FcεRI on mast cells and CD23 on B cells, are mediated by the Fc region, a dimer of the Cε2, Cε3 and Cε4 domains. A sub-fragment lacking the Cε2 domains, Fcε3–4, also binds to both receptors, although receptor binding almost exclusively involves the Cε3 domains. This domain also contains the N-linked glycosylation site conserved in other isotypes. We report here the crystal structures of IgE-Fc and Fcε3–4 at the highest resolutions yet determined, 1.75 Å and 2.0 Å respectively, revealing unprecedented detail regarding the carbohydrate and its interactions with protein domains. Analysis of the crystallographic B factors of these, together with all earlier IgE-Fc and Fcε3–4 structures, shows that the Cε3 domains exhibit the greatest intrinsic flexibility and quaternary structural variation within IgE-Fc. Intriguingly, both well-ordered carbohydrate and disordered polypeptide can be seen within the same Cε3 domains. A simplified method for comparing the quaternary structures of the Cε3 domains in free and receptor-bound IgE-Fc structures is presented, which clearly delineates the FcεRI and CD23 bound states. Importantly, differential scanning fluorimetric analysis of IgE-Fc and Fcε3–4 identifies Cε3 as the domain most susceptible to thermally-induced unfolding, and responsible for the characteristically low melting temperature of IgE.
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Aug 2017
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
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Anna M.
Davies
,
Elizabeth G.
Allan
,
Anthony H.
Keeble
,
Jean
Delgado
,
Benjamin P.
Cossins
,
Alkistis N.
Mitropoulou
,
Marie O. Y.
Pang
,
Tom
Ceska
,
Andrew J.
Beavil
,
Graham
Craggs
,
Marta
Westwood
,
Alistair J.
Henry
,
James M.
Mcdonnell
,
Brian J.
Sutton
Diamond Proposal Number(s):
[1220]
Open Access
Abstract: Immunoglobulin E and its interactions with receptors FcϵRI and CD23 play a central role in allergic disease. Omalizumab, a clinically-approved therapeutic antibody, inhibits the interaction between IgE and FcϵRI, preventing mast cell and basophil activation, and blocks IgE binding to CD23 on B cells and antigen-presenting cells. We solved the crystal structure of the complex between an omalizumab-derived Fab and IgE-Fc, with one Fab bound to each Cϵ3 domain. Free IgE-Fc adopts an acutely bent structure, but in the complex it is only partially bent, with large-scale conformational changes in the Cϵ3 domains that inhibit the interaction with FcϵRI. CD23 binding is inhibited sterically due to overlapping binding sites on each Cϵ3 domain. Studies of omalizumab Fab binding in solution demonstrate the allosteric basis for FcϵRI inhibition and, together with the structure, reveal how omalizumab may accelerate dissociation of receptor-bound IgE from FcϵRI, exploiting the intrinsic flexibility and allosteric potential of IgE.
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Apr 2017
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9495]
Open Access
Abstract: The antibody IgE plays a central role in allergic disease mechanisms. Its effector functions are controlled through interactions between the Fc region and two principal cell surface receptors FcεRI and CD23. The interaction with FcεRI is primarily responsible for allergic sensitization and the inflammatory response, while IgE binding to CD23 is involved in the regulation of IgE synthesis and allergen transcytosis. Here we present the crystal structure of a CD23/IgE-Fc complex and conduct isothermal titration calorimetric binding studies. Two lectin-like “head” domains of CD23 bind to IgE-Fc with affinities that differ by more than an order of magnitude, but the crystal structure reveals only one head bound to one of the two identical heavy-chains in the asymmetrically bent IgE-Fc. These results highlight the subtle interplay between receptor binding sites in IgE-Fc and their affinities, the understanding of which may be exploited for therapeutic intervention in allergic disease.
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Mar 2017
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I03-Macromolecular Crystallography
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Nyssa
Drinkwater
,
Benjamin P.
Cossins
,
Anthony H.
Keeble
,
Michael
Wright
,
Katharine
Cain
,
Hanna
Hailu
,
Amanda
Oxbrow
,
Jean
Delgado
,
Lindsay K
Shuttleworth
,
Michael W.-P.
Kao
,
James M.
Mcdonnell
,
Andrew J.
Beavil
,
Alistair J.
Henry
,
Brian J.
Sutton
Abstract: Crystallographic and solution studies have shown that IgE molecules are acutely bent in their Fc region. Crystal structures reveal the Cɛ2 domain pair folded back onto the Cɛ3-Cɛ4 domains, but is the molecule exclusively bent or can the Cɛ2 domains adopt extended conformations and even 'flip' from one side of the molecule to the other? We report the crystal structure of IgE-Fc captured in a fully extended, symmetrical conformation and show by molecular dynamics, calorimetry, stopped-flow kinetic, surface plasmon resonance (SPR) and Förster resonance energy transfer (FRET) analyses that the antibody can indeed adopt such extended conformations in solution. This diversity of conformational states available to IgE-Fc offers a new perspective on IgE function in allergen recognition, as part of the B-cell receptor and as a therapeutic target in allergic disease.
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Mar 2014
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: IgE antibodies play a central role in allergic disease. They recognize allergens via their Fab regions, whilst their effector functions are controlled through interactions of the Fc region with two principal cell surface receptors, FcɛRI and CD23. Crosslinking of FcɛRI-bound IgE on mast cells and basophils by allergen initiates an immediate inflammatory response, while the interaction of IgE with CD23 on B-cells regulates IgE production. We have determined the structures of the C-type lectin “head” domain of CD23 from seven crystal forms. The thirty-five independent structures reveal extensive conformational plasticity in two loops that are critical for IgE binding.
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Dec 2013
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Open Access
Abstract: Immunoglobulin E (IgE) antibodies play a fundamental role in allergic disease and are a target for therapeutic intervention. IgE functions principally through two receptors, Fc?RI and CD23 (Fc?RII). Minute amounts of allergen trigger mast cell or basophil degranulation by cross-linking IgE-bound Fc?RI, leading to an inflammatory response. The interaction between IgE and CD23 on B-cells regulates IgE synthesis. CD23 is unique among Ig receptors in that it belongs to the C-type (calcium-dependent) lectin-like superfamily. Although the interaction of CD23 with IgE is carbohydrate-independent, calcium has been reported to increase the affinity for IgE, but the structural basis for this activity has previously been unknown. We have determined the crystal structures of the human lectin-like head domain of CD23 in its Ca2+-free and Ca2+-bound forms, as well as the crystal structure of the Ca2+-bound head domain of CD23 in complex with a subfragment of IgE-Fc consisting of the dimer of C?3 and C?4 domains (Fc?3-4). Together with site-directed mutagenesis, the crystal structures of four Ca2+ ligand mutants, isothermal titration calorimetry, surface plasmon resonance, and stopped-flow analysis, we demonstrate that Ca2+ binds at the principal and evolutionarily conserved binding site in CD23. Ca2+ binding drives Pro-250, at the base of an IgE-binding loop (loop 4), from the trans to the cis configuration with a concomitant conformational change and ordering of residues in the loop. These Ca2+-induced structural changes in CD23 lead to additional interactions with IgE, a more entropically favorable interaction, and a 30-fold increase in affinity of a single head domain of CD23 for IgE. Taken together, these results suggest that binding of Ca2+ brings an extra degree of modulation to CD23 function.
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Jun 2013
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I04-Macromolecular Crystallography
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Abstract: Antibodies of the human IgG4 subclass uniquely undergo a process of Fab-arm exchange in which the heavy-chains of antibodies of different specificities can dissociate and then recombine. The mechanism by which the resulting functionally monovalent but bi-specific antibodies are formed is not only key to understanding their biological role, but is also important for the design of therapeutic monoclonal antibodies. Both the hinge region and the C(H)3 domain interface are known to be involved, and of the residues that differ between human IgG1 and IgG4 in C(H)3, residue 409, the only difference at the interface itself, has been implicated. We report the high resolution (1.8 Angstrom) structure of the C(H)3 domain dimer of IgG4, and find that Arg409 in IgG4, when compared with Lys409 observed in high resolution IgG1 structures, disrupts a network of water-mediated hydrogen bonding that is conserved in IgG1. Other conformational differences were detected that are a consequence of the presence of Arg409, such as a widening of the separation between residues Asn390 in one domain and Ser 400 in the other, which opens up a groove at the edge of the interface in IgG4 compared with IgG1. The effect of all these differences on the C(H)3 interface, doubled as a result of the interface's two-fold symmetry, is weakening of the inter-domain interaction in IgG4 compared with IgG1. This suggests a mechanism by which Arg409 weakens the C(H)3 interface in IgG4, predisposing this human antibody subclass to Fab-arm exchange.
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Nov 2012
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