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A Fluorescent Biosensor Reveals Conformational Changes in Human Immunoglobulin E Fc

IMPLICATIONS FOR MECHANISMS OF RECEPTOR BINDING, INHIBITION, AND ALLERGEN RECOGNITION*
  • James Hunt
    Footnotes
    Affiliations
    MRC and Asthma UK Centre in Allergic Mechanisms of Asthma, Guy's Hospital Campus, London SE1 1UL

    The Randall Division of Cell and Molecular Biophysics, Guy's Hospital Campus, London SE1 1UL

    The Division of Asthma Allergy and Lung Biology, King's College London, Guy's Hospital Campus, London SE1 1UL
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  • Anthony H. Keeble
    Affiliations
    MRC and Asthma UK Centre in Allergic Mechanisms of Asthma, Guy's Hospital Campus, London SE1 1UL

    The Randall Division of Cell and Molecular Biophysics, Guy's Hospital Campus, London SE1 1UL

    The Division of Asthma Allergy and Lung Biology, King's College London, Guy's Hospital Campus, London SE1 1UL
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  • Robert E. Dale
    Affiliations
    The Randall Division of Cell and Molecular Biophysics, Guy's Hospital Campus, London SE1 1UL
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  • Melissa K. Corbett
    Affiliations
    The Randall Division of Cell and Molecular Biophysics, Guy's Hospital Campus, London SE1 1UL
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  • Rebecca L. Beavil
    Affiliations
    MRC and Asthma UK Centre in Allergic Mechanisms of Asthma, Guy's Hospital Campus, London SE1 1UL

    The Randall Division of Cell and Molecular Biophysics, Guy's Hospital Campus, London SE1 1UL

    The Division of Asthma Allergy and Lung Biology, King's College London, Guy's Hospital Campus, London SE1 1UL
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  • James Levitt
    Affiliations
    The Department of Physics, King's College London, Strand, London WC2R 2LS
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  • Marcus J. Swann
    Affiliations
    Farfield Group Limited, Voyager, Chicago Avenue, Manchester Airport, Manchester, M90 3DQ, United Kingdom
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  • Klaus Suhling
    Affiliations
    The Department of Physics, King's College London, Strand, London WC2R 2LS
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  • Simon Ameer-Beg
    Affiliations
    The Randall Division of Cell and Molecular Biophysics, Guy's Hospital Campus, London SE1 1UL
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  • Brian J. Sutton
    Affiliations
    MRC and Asthma UK Centre in Allergic Mechanisms of Asthma, Guy's Hospital Campus, London SE1 1UL

    The Randall Division of Cell and Molecular Biophysics, Guy's Hospital Campus, London SE1 1UL
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  • Andrew J. Beavil
    Correspondence
    To whom correspondence should be addressed: King's College London, Randall Division of Cell and Molecular Biophysics, New Hunt's House, Guy's Campus, London SE1 1UL, UK. Tel.: 44-20-78488064; Fax: 44-20-78486435
    Affiliations
    MRC and Asthma UK Centre in Allergic Mechanisms of Asthma, Guy's Hospital Campus, London SE1 1UL

    The Randall Division of Cell and Molecular Biophysics, Guy's Hospital Campus, London SE1 1UL

    The Division of Asthma Allergy and Lung Biology, King's College London, Guy's Hospital Campus, London SE1 1UL
    Search for articles by this author
  • Author Footnotes
    * This work was supported by a grant from Asthma UK and the Medical Research Council (UK).
    This article contains supplemental information, Tables S1–S4, Figs. S1–S11, Equations S1–S20, and a video.
    1 Present address: Novartis Institutes for Biomedical Research, Wimblehurst Rd., Horsham, West Sussex, RH12 5AB, UK.
Open AccessPublished:March 22, 2012DOI:https://doi.org/10.1074/jbc.M111.331967
      IgE binding to its high affinity receptor FcϵRI on mast cells and basophils is a key step in the mechanism of allergic disease and a target for therapeutic intervention. Early indications that IgE adopts a bent structure in solution have been confirmed by recent x-ray crystallographic studies of IgEFc, which further showed that the bend, contrary to expectation, is enhanced in the crystal structure of the complex with receptor. To investigate the structure of IgEFc and its conformational changes that accompany receptor binding in solution, we created a Förster resonance energy transfer (FRET) biosensor using biologically encoded fluorescent proteins fused to the N- and C-terminal IgEFc domains (Cϵ2 and Cϵ4, respectively) together with the theoretical basis for quantitating its behavior. This revealed not only that the IgEFc exists in a bent conformation in solution but also that the bend is indeed enhanced upon FcϵRI binding. No change in the degree of bending was seen upon binding to the B cell receptor for IgE, CD23 (FcϵRII), but in contrast, binding of the anti-IgE therapeutic antibody omalizumab decreases the extent of the bend, implying a conformational change that opposes FcϵRI engagement. HomoFRET measurements further revealed that the (Cϵ2)2 and (Cϵ4)2 domain pairs behave as rigid units flanking the conformational change in the Cϵ3 domains. Finally, modeling of the accessible conformations of the two Fab arms in FcϵRI-bound IgE revealed a mutual exclusion not seen in IgG and Fab orientations relative to the membrane that may predispose receptor-bound IgE to cross-linking by allergens.

      Introduction

      The incidence of allergic disease is on the increase world-wide, and this includes a range of conditions from seasonal hay fever to asthma and fatal anaphylactic shock triggered by allergens such as peanuts. The key mediator between the allergen and the cells of the immune system is the antibody immunoglobulin E (IgE) (
      • Gould H.J.
      • Sutton B.J.
      IgE in allergy and asthma today.
      ). In contrast to the comparatively high levels of the other immunoglobulin isotypes (∼2 mg/ml for IgA and 13 mg/ml for IgG), IgE is only found at low levels (∼50–200 ng/ml) in serum. Nevertheless, IgE binds with high affinity (Ka ∼ 1010 m−1) through its Fc region to the receptor FcϵRI expressed on the surface of mast cells and basophils, and this high affinity and long lifetime of receptor-bound IgE in tissue (dissociation t½ ∼14 days) (
      • Wank S.A.
      • DeLisi C.
      • Metzger H.
      Analysis of the rate-limiting step in a ligand-cell receptor interaction. The IgE system.
      ,
      • Miller L.
      • Blank U.
      • Metzger H.
      • Kinet J.
      Expression of high affinity binding of human immunoglobulin E by transfected cells.
      ) accounts for the allergic sensitization and rapid activation of these cells upon allergen binding. Degranulation of these cells and the release of inflammatory mediators, triggered by often innocuous allergens, leads to the physiological responses associated with allergic reactions (
      • Gould H.J.
      • Sutton B.J.
      IgE in allergy and asthma today.
      ,
      • Gould H.J.
      • Sutton B.J.
      • Beavil A.J.
      • Beavil R.L.
      • McCloskey N.
      • Coker H.A.
      • Fear D.
      • Smurthwaite L.
      The biology of IgE and the basis of allergic disease.
      ), although the primary function of IgE may be to respond to multicellular parasitic pathogens such as helminths (
      • Gounni A.S.
      • Lamkhioued B.
      • Ochiai K.
      • Tanaka Y.
      • Delaporte E.
      • Capron A.
      • Kinet J.P.
      • Capron M.
      High affinity IgE receptor on eosinophils is involved in defense against parasites.
      ).
      In IgG, the two Fab (antigen binding) arms are connected to the Fc (a dimer of Cγ2 and Cγ3 domains) by a disulfide-linked hinge region. IgE, however, has the (Cϵ2)2 domain pair in place of the hinge, and thus IgEFc comprises six domains (a dimer of Cϵ2, Cϵ3, and Cϵ4, disulfide-linked between the Cϵ2 domains). The crystal structure of IgEFc revealed the (Cϵ2)2 domain pair bent back against the Cϵ3 domains and even contacted one of the Cϵ4 domains (
      • Wan T.
      • Beavil R.L.
      • Fabiane S.M.
      • Beavil A.J.
      • Sohi M.K.
      • Keown M.
      • Young R.J.
      • Henry A.J.
      • Owens R.J.
      • Gould H.J.
      • Sutton B.J.
      The crystal structure of IgE Fc reveals an asymmetrically bent conformation.
      ). This structure was even more acutely bent than that proposed in an earlier FRET study with a chimeric IgE (
      • Zheng Y.
      • Shopes B.
      • Holowka D.
      • Baird B.
      Conformations of IgE bound to its receptor FcϵRI and in solution.
      ) or an x-ray and neutron scattering study of IgEFc in solution (
      • Beavil A.J.
      • Young R.J.
      • Sutton B.J.
      • Perkins S.J.
      Bent domain structure of recombinant human IgE-Fc in solution by X-ray and neutron scattering in conjunction with an automated curve-fitting procedure.
      ). Comparative studies of the kinetics of binding to the soluble extracellular domains of the IgE-binding α-chain of FcϵRI (sFcϵRIα)
      The abbreviations used are: sFcϵRIα
      soluble fragment of the high affinity IgE receptor FcϵRI α-chain
      mRFP
      monomeric red fluorescent protein
      FP
      fluorescent protein
      eGFP
      enhanced green fluorescent protein.
      of both IgEFc and a subfragment lacking the Cϵ2 domains (IgEFc3–4) demonstrated that the Cϵ2 domains were in part responsible for the high affinity and slow dissociation rate (
      • McDonnell J.M.
      • Calvert R.
      • Beavil R.L.
      • Beavil A.J.
      • Henry A.J.
      • Sutton B.J.
      • Gould H.J.
      • Cowburn D.
      The structure of the IgE Cϵ2 domain and its role in stabilizing the complex with its high affinity receptor FcϵRIα.
      ). Although an “unbending” of the IgEFc was proposed to allow engagement of the Cϵ2 domains with FcϵRI (
      • Gould H.J.
      • Sutton B.J.
      • Beavil A.J.
      • Beavil R.L.
      • McCloskey N.
      • Coker H.A.
      • Fear D.
      • Smurthwaite L.
      The biology of IgE and the basis of allergic disease.
      ), IgEFc was found to remain bent (in fact the bend is enhanced) in the crystal structure of the IgEFc·sFcϵRIα complex (
      • Holdom M.D.
      • Davies A.M.
      • Nettleship J.E.
      • Bagby S.C.
      • Dhaliwal B.
      • Girardi E.
      • Hunt J.
      • Gould H.J.
      • Beavil A.J.
      • McDonnell J.M.
      • Owens R.J.
      • Sutton B.J.
      Conformational changes in IgE contribute to its uniquely slow dissociation rate from receptor FcϵRI.
      ).
      To discover whether the conformations and conformational changes seen in the crystal structures occur in solution, we generated an IgEFc biosensor to monitor conformational change in this region of the molecule. Previous solution-based fluorescence depolarization studies of human and murine IgE provided evidence only of flexibility of the Fabs relative to Fc (
      • Oi V.T.
      • Vuong T.M.
      • Hardy R.
      • Reidler J.
      • Dangle J.
      • Herzenberg L.A.
      • Stryer L.
      Correlation between segmental flexibility and effector function of antibodies.
      ,
      • Slattery J.
      • Holowka D.
      • Baird B.
      Segmental flexibility of receptor-bound immunoglobulin E.
      ,
      • Holowka D.
      • Wensel T.
      • Baird B.
      A nanosecond fluorescence depolarization study on the segmental flexibility of receptor-bound immunoglobulin E.
      ); this was also observed by electron microscopy (
      • Roux K.H.
      • Strelets L.
      • Brekke O.H.
      • Sandlie I.
      • Michaelsen T.E.
      Comparisons of the ability of human IgG3 hinge mutants, IgM, IgE, and IgA2, to form small immune complexes. A role for flexibility and geometry.
      ). FRET studies using recombinant mouse chimeric IgE (with Cϵ4 replaced by Cγ3 of IgG) did reveal a small change in energy transfer on binding to rat FcϵRI (
      • Zheng Y.
      • Shopes B.
      • Holowka D.
      • Baird B.
      Conformations of IgE bound to its receptor FcϵRI and in solution.
      ,
      • Zheng Y.
      • Shopes B.
      • Holowka D.
      • Baird B.
      Dynamic conformations compared for IgE and IgG1 in solution and bound to receptors.
      ), but the donor fluorophore was placed in the Fab antigen binding site rather than in Fc, making it difficult to distinguish between movements within Fc from those of the Fabs relative to Fc. We report here a conformational change within human IgEFc monitored by FRET between biologically encoded probes attached to the N and C termini of the molecule. Both hetero- and homo-energy transfer were measured using a combination of steady-state and excited-state decay techniques. These measurements were made in the presence and absence of soluble forms of the “high affinity” mast cell receptor FcϵRIα and the “low affinity” B cell receptor CD23 (FcϵRII) as well as the Fab of the therapeutic anti-IgE antibody omalizumab (XolairTM). The results reveal not only that IgEFc is bent in solution but also that the bend is enhanced upon binding to FcϵRIα. CD23 binding has no effect upon the bend, whereas the anti-IgE omalizumab causes an unbending. Finally, we modeled the disposition of the Fabs relative to FcϵRI-bound IgEFc and discovered a significant difference compared with receptor-bound IgG, which may have important implications for cross-linking of receptor-bound IgE and the activation of mast cells and basophils by allergens.

      DISCUSSION

      Previous efforts to investigate IgE flexibility and conformation focused on the whole antibody. Anisotropy decay measurements on fluorescently labeled murine IgE revealed less flexibility than IgG (
      • Oi V.T.
      • Vuong T.M.
      • Hardy R.
      • Reidler J.
      • Dangle J.
      • Herzenberg L.A.
      • Stryer L.
      Correlation between segmental flexibility and effector function of antibodies.
      ). Steady-state fluorescence anisotropy data obtained using the same murine IgE showed no change in flexibility on FcϵRI binding (
      • Slattery J.
      • Holowka D.
      • Baird B.
      Segmental flexibility of receptor-bound immunoglobulin E.
      ), although later time-resolved analyses distinguished two components, one that was unaffected by receptor binding and abrogated when the Fab arms were cross-linked, and another, longer time-scale movement that was affected by receptor binding (
      • Holowka D.
      • Wensel T.
      • Baird B.
      A nanosecond fluorescence depolarization study on the segmental flexibility of receptor-bound immunoglobulin E.
      ). Together with FRET measurements (
      • Baird B.
      • Holowka D.
      Structural mapping of Fc receptor-bound immunoglobulin E. Proximity to the membrane surface of the antibody combining site and another site in the Fab segments.
      ), this implied a rigidly bent IgE molecule bound to FcϵRI with “wagging” Fabs directed away from the cell surface (
      • Holowka D.
      • Wensel T.
      • Baird B.
      A nanosecond fluorescence depolarization study on the segmental flexibility of receptor-bound immunoglobulin E.
      ). In further studies with a chemically FRET-labeled chimeric murine IgE/IgG, a bent conformation was confirmed that became less flexible and slightly more bent when bound to FcϵRI on cells (
      • Zheng Y.
      • Shopes B.
      • Holowka D.
      • Baird B.
      Conformations of IgE bound to its receptor FcϵRI and in solution.
      ,
      • Zheng Y.
      • Shopes B.
      • Holowka D.
      • Baird B.
      Dynamic conformations compared for IgE and IgG1 in solution and bound to receptors.
      ). This was also consistent with the “cusp-like” structure of rat myeloma IgE inferred from x-ray solution scattering (
      • Davis K.G.
      • Glennie M.
      • Harding S.E.
      • Burton D.R.
      A model for the solution conformation of rat IgE.
      ). A compact structure for human IgEFc was also shown by solution scattering (
      • Beavil A.J.
      • Young R.J.
      • Sutton B.J.
      • Perkins S.J.
      Bent domain structure of recombinant human IgE-Fc in solution by X-ray and neutron scattering in conjunction with an automated curve-fitting procedure.
      ), and the crystal structures of both free (
      • Wan T.
      • Beavil R.L.
      • Fabiane S.M.
      • Beavil A.J.
      • Sohi M.K.
      • Keown M.
      • Young R.J.
      • Henry A.J.
      • Owens R.J.
      • Gould H.J.
      • Sutton B.J.
      The crystal structure of IgE Fc reveals an asymmetrically bent conformation.
      ) and receptor-bound (
      • Holdom M.D.
      • Davies A.M.
      • Nettleship J.E.
      • Bagby S.C.
      • Dhaliwal B.
      • Girardi E.
      • Hunt J.
      • Gould H.J.
      • Beavil A.J.
      • McDonnell J.M.
      • Owens R.J.
      • Sutton B.J.
      Conformational changes in IgE contribute to its uniquely slow dissociation rate from receptor FcϵRI.
      ) IgEFc reveal asymmetrically and acutely bent conformations, with a more acute bend in the complex.
      In the present study we show that the asymmetrically bent IgEFc conformation exists in solution by quantitatively modeling heteroFRET and homoFRET energy transfer and depolarization processes within an ensemble of 1300 models. Rigid-body simulations that sample the conformational space of the fluorescent protein (FP) domains within the IgEFc biosensor allowed detailed calculation of distances between the FRET partners as well as their relative orientations. These factors enabled us to determine for each conformer the degree of depolarization in homoFRET and to avoid inappropriately assuming the problematic 2/3 value for orientation factors (Equation 10) needed in both heteroFRET and homoFRET calculations. This assures optimally reliable predictions of the population average heteroFRET efficiency in the mRFP-IgEFc-eGFP construct and of the population average homoFRET-induced fluorescence anisotropy decays of the C-terminal and the N-terminal eGFP constructs, to compare with the experimental measurements. A remarkable degree of correspondence is found.
      Upon binding to sFcϵRIα, the FRET efficiency of the mRFP-IgEFc-eGFP biosensor increases, as measured by a decrease in the average eGFP fluorescence lifetime, suggesting that the bent structure of the IgEFc becomes more bent upon sFcϵRIα binding in solution (Equation 8). Increased bending of IgEFc within the IgEFc·sFcϵRIα complex was also observed by crystallography (
      • Holdom M.D.
      • Davies A.M.
      • Nettleship J.E.
      • Bagby S.C.
      • Dhaliwal B.
      • Girardi E.
      • Hunt J.
      • Gould H.J.
      • Beavil A.J.
      • McDonnell J.M.
      • Owens R.J.
      • Sutton B.J.
      Conformational changes in IgE contribute to its uniquely slow dissociation rate from receptor FcϵRI.
      ), and we show this is not an artifact of crystal packing. Additionally, the homoFRET results show that during this conformational change the (Cϵ2)2 and the (Cϵ4)2 domain pairs appear to behave as rigid units.
      The IgEFc biosensor undergoes no measurable change in FRET efficiency upon binding to CD23 (FcϵRII). Two molecules of derCD23 (the IgE-binding lectin domain) can bind to IgEFc (
      • Shi J.
      • Ghirlando R.
      • Beavil R.L.
      • Beavil A.J.
      • Keown M.B.
      • Young R.J.
      • Owens R.J.
      • Sutton B.J.
      • Gould H.J.
      Interaction of the low affinity receptor CD23/FcϵRII lectin domain with the Fcϵ3-4 fragment of human immunoglobulin E.
      ) at locations that are distinct from the FcϵRI binding site (as indicated by mutagenesis (
      • Sayers I.
      • Housden J.E.
      • Spivey A.C.
      • Helm B.A.
      The importance of Lys-352 of human immunoglobulin E in FcϵRII/CD23 recognition.
      )). Clearly the binding mechanisms also differ, and IgEFc can bind to derCD23 in its naturally bent conformation without an apparent measurable change in the bend angle.
      The binding of the Fab fragment of omalizumab (an anti-IgE therapeutic IgG) caused a decrease in the FRET signal (as measured by an increase in the average donor fluorescence lifetime), indicative of an unbending of the IgEFc. Again, the precise location of the epitope recognized by omalizumab is not known, but it acts by preventing the binding of free IgE molecules to FcϵRI (
      • Holgate S.
      • Casale T.
      • Wenzel S.
      • Bousquet J.
      • Deniz Y.
      • Reisner C.
      The anti-inflammatory effects of omalizumab confirm the central role of IgE in allergic inflammation.
      ). This mechanism may thus be indirect and allosteric, as a result of bending the IgE in the opposite direction to that required for receptor engagement.
      Changes in FRET efficiency could potentially arise for reasons other than changes in inter-probe separation, but these can be discounted. (i) sFcϵRIα binding could distort the eGFP fluorophore and/or its close environment altering the distribution between its different photophysical states, so that the average lifetime changes for a reason other than FRET. This appears unlikely, however, as sFcϵRIα binding has no effect on the donor alone control (see supplemental Table 3). (ii) A structural change in the eGFP protein could lead to a change in the steady-state spectra (
      • Kirchhofer A.
      • Helma J.
      • Schmidthals K.
      • Frauer C.
      • Cui S.
      • Karcher A.
      • Pellis M.
      • Muyldermans S.
      • Casas-Delucchi C.S.
      • Cardoso M.C.
      • Leonhardt H.
      • Hopfner K.P.
      • Rothbauer U.
      Modulation of protein properties in living cells using nanobodies.
      ), but this is not observed upon sFcϵRIα binding (Fig. 1D). Furthermore, the modeling also showed no steric clashes between sFcϵRIα and the FP domains. (iii) The change in FRET efficiency could be solely due to a change in the relative orientations of the FPs in the IgEFc without any appreciable change in distances between them. This can be discounted, first because an unfeasibly large (30–40%) change in the population average κ2 would be required, in contrast to only a 4–5% change in distance. Second, and perhaps more importantly, other experimental evidence indicates that the distribution of orientations of the fluorescent protein probes is unchanged upon sFcϵRIα binding. This is provided by steady-state fluorescence anisotropy (Fig. 3, C and D) and anisotropy decay measurements (supplemental Fig. 8, B and C), where no change is observed. Furthermore, analysis of the biosensor models showed that there was a close to random distribution of κ2 irrespective of the interprobe distance (see supplemental Figs. S6 and S7), suggesting that even if the space available to the probes is more constrained upon sFcϵRIα binding, the randomness of their orientations is not likely to be affected.
      The good agreement between the observed FRET efficiencies and those calculated from the modeled IgEFc biosensor suggested that the flexibility of the Fab arms might also be modeled reliably. A comparison between the range of conformations available to the Fab arms in IgE and IgG revealed marked differences (Fig. 4, supplemental video). In IgG, not only do the regions accessible to the two Fab arms overlap extensively, but when mapped onto the crystal structure of an IgG·FcγRIII complex there is considerable clashing with the receptor and also with the membrane. Remarkably, the Fab arms of IgE occupy distinct regions (Fig. 4A) and show virtually no clashing with either the receptor or membrane. The rigid (Cϵ2)2 domain pair appears to act as a steric insulator and presents (and conformationally restricts) the Fab arms in a manner unique to IgE. In part, this difference between IgG and IgE may relate to a difference in biological function; IgG·antigen complexes generally form in solution and then interact with their low affinity receptors, whereas most IgE is receptor-bound before encountering antigen (allergen). If IgE had an IgG-like structure, the Fabs would be much closer to both the cell membrane and the receptor, which might inhibit antigen recognition. However, the manner in which the bent IgE molecule and its (Cϵ2)2 domains present the Fab arms may have important implications for allergen recognition and effector cell activation.
      The crystal structure of the high affinity FcγRI, which has an additional domain compared with FcγRII and FcγRIII, is instructive in this context. The biological roles of the high affinity IgG·FcγRI and IgE·FcϵRI complexes have common features; 1) they are both found on dendritic cells where the high affinity results in their action as scavenger receptors that bind and internalize antigens; 2) they are both found on cells that combat infecting organisms; that is, macrophages and bacterial infections for IgG·FcγRI and mast cells and helminth infections for IgE·FcϵRI. The FcγRI crystal structure (PDB code 3RJD (
      • Lu J.
      • Ellsworth J.L.
      • Hamacher N.
      • Oak S.W.
      • Sun P.D.
      Crystal structure of Fcγ receptor I and its implication in high affinity γ-immunoglobulin binding.
      )) shows that the additional extracellular domain (D3), despite the predictions from mutagenesis (
      • Harrison P.T.
      • Allen J.M.
      High affinity IgG binding by FcγRI (CD64) is modulated by two distinct IgSF domains and the transmembrane domain of the receptor.
      ), does not contact IgG. Instead, it acts as a spacer pushing the D1 and D2 domains (that do contact IgG) away from the membrane (Fig. 4B). Therefore, it is a similar adaptation to the Cϵ2 domains in IgE (only this time in the receptor) that allows the Fabs to move freely without clashing with the membrane, an important ability for a scavenging receptor.
      As depicted in Fig. 4A, one of the IgE Fabs predominantly points away from the membrane, whereas the other is oriented parallel to the membrane. The optimal inter-IgE epitope distance for FcϵRI activation has been shown to be 40–60 Å using rigid dinitrophenol-labeled molecular spacers (
      • Paar J.M.
      • Harris N.T.
      • Holowka D.
      • Baird B.
      Bivalent ligands with rigid double-stranded DNA spacers reveal structural constraints on signaling by FcϵRI.
      ,
      • Sil D.
      • Lee J.B.
      • Luo D.
      • Holowka D.
      • Baird B.
      Trivalent ligands with rigid DNA spacers reveal structural requirements for IgE receptor signaling in RBL mast cells.
      ). Distances shorter than this were not assayed, but the efficiency of activation decreased rapidly at greater separations. A key feature of allergens is the spatial clustering of more than one IgE epitope and for small allergens or those presenting only a single epitope such as β-lactoglobulin (Bos d 5), this frequently occurs through oligomerization. Two crystal structures of dimerized allergens with bound Fabs have been solved, Bos d 5 (2R56 (
      • Niemi M.
      • Jylhä S.
      • Laukkanen M.L.
      • Söderlund H.
      • Mäkinen-Kiljunen S.
      • Kallio J.M.
      • Hakulinen N.
      • Haahtela T.
      • Takkinen K.
      • Rouvinen J.
      Molecular interactions between a recombinant IgE antibody and the β-lactoglobulin allergen.
      ) and Bla g 2 (PDB code 2NR6 (
      • Li M.
      • Gustchina A.
      • Alexandratos J.
      • Wlodawer A.
      • Wünschmann S.
      • Kepley C.L.
      • Chapman M.D.
      • Pomés A.
      Crystal structure of a dimerized cockroach allergen Bla g 2 complexed with a monoclonal antibody.
      )) and are both shown in Fig. 4C. These two complexes are particularly instructive for not only do they reveal that the allergen epitopes are optimally spaced for receptor activation (40 and 63 Å, respectively), but they also both display an approximately co-linear arrangement, consistent with IgE Fabs on adjacent receptors oriented parallel to the membrane. The acutely bent receptor-bound IgEFc and conformationally constrained Fab arms may, therefore, predispose IgE-sensitized cells to respond to small antigens with only a particular arrangement of epitopes; this may represent a structural determinant of protein allergenicity, the understanding of which has hitherto proved elusive.

      Acknowledgments

      We thank members of the Sutton and Beavil groups past and present for useful discussions. S.A-B acknowledges his collaborators in the development of the TRI2 software.

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