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Human immunoglobulin E flexes between acutely bent and extended conformations

Nature Structural & Molecular Biology volume 21, pages 397–404 (2014)Cite this article

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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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Figure 1: Bent and extended structures adopted by IgE-Fc.
Figure 2: Molecular dynamics simulation of IgE-Fc unbending.
Figure 3: ITC and FRET analyses of aɛFab binding to IgE-Fc.
Figure 4: Proposed mechanism of IgE-Fc flexibility and aɛFab binding in solution.
Figure 5: Structural basis for inhibition of IgE-Fc interaction with FcɛRIα by aɛFab.
Figure 6: Modeled structure of the entire IgE molecule in different biological contexts.

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Acknowledgements

The authors thank the Medical Research Council UK (G1100090; B.J.S.) and The Wellcome Trust for grant funding (076343; B.J.S.) and support for the King's Biomolecular Spectroscopy Centre (085944). The work was carried out with the support of Asthma UK, the National Institute for Health Research Biomedical Research Center and the Diamond Light Source (Harwell, UK).

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  1. Nyssa Drinkwater and Benjamin P Cossins: These authors contributed equally to this work.

Authors and Affiliations

  1. Randall Division of Cell and Molecular Biophysics, King's College London, London, UK

    Nyssa Drinkwater, Anthony H Keeble, Michael W-P Kao, James M McDonnell, Andrew J Beavil & Brian J Sutton

  2. Medical Research Council and Asthma UK Centre in Allergic Mechanisms of Asthma, London, UK

    Nyssa Drinkwater, Anthony H Keeble, Michael W-P Kao, James M McDonnell, Andrew J Beavil & Brian J Sutton

  3. UCB Pharma, Slough, UK

    Benjamin P Cossins, Michael Wright, Katharine Cain, Hanna Hailu, Amanda Oxbrow, Jean Delgado, Lindsay K Shuttleworth & Alistair J Henry

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  1. Nyssa Drinkwater
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Contributions

N.D. and B.P.C. contributed equally to this work. N.D. performed the crystallography and structure analysis, N.D. and J.M.M. conducted SPR experiments, and N.D. and B.J.S. wrote the manuscript. B.C. undertook the molecular dynamics and contributed to writing. A.H.K. was responsible for the ITC and stopped-flow analyses, M.W. generated the aɛFab molecule, K.C. performed antibody engineering, H.H. expressed the proteins, A.O. purified the proteins, J.D. and L.K.S. collected intramolecular FRET data using reagents made by M.W.-P.K., and A.J.H. J.M.M., A.J.H. and A.J.B. contributed to writing, data interpretation. B.J.S. designed and supervised the research.

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Correspondence to Brian J Sutton.

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Integrated supplementary information

Supplementary Figure 1 Conformational flexibility of the Cɛ3 domains.

(a) Directions of “open/closed” and “swing” movements between the Cɛ3 domains are indicated on the free IgE-Fc structure (2WQR, Cɛ2 domains not shown for clarity). IgE-FcA is shown in blue, IgE-FcB in orange. (b) Conformational change of the Cɛ3 domains of IgE-Fc upon aɛFab binding. The Cɛ3 and Cɛ4 domains of the extended IgE-Fc structure as seen in the aɛFab complex (IgE-FcA in blue, IgE-FcB in orange) are overlaid on the Cɛ3 and Cɛ4 domains of free IgE-Fc (grey) (Cɛ2 domains not shown for clarity). In the structure of free IgE-Fc, IgE-FcA is in the closed conformation, and IgE-FcB is in the open conformation, while in the extended IgE-Fc structure, both chains are open. Open (extended IgE-Fc) and closed (free IgE-Fc) forms of IgE-FcA are indicated.

Supplementary Figure 2 Interactions between aɛFab and IgE-Fc.

(a) Interactions between aɛFab1 heavy chain (green) and the Cɛ2 domain of IgE-FcA (blue). Hydrogen bonds are indicated by black lines. (b) Contact between IgE-Fc Cɛ2-Cɛ3 linker regions and the aɛFab molecules. IgE-FcA is shown in blue and IgE-FcB in orange; aɛFab1 and aɛFab2 are shown in green. The locations of the Cɛ2 and Cɛ3 domains are indicated. (c) R393 binding pocket between aɛFab heavy (green) and light (grey) chains. Black lines indicate hydrogen bonds formed with aɛFab residues. (d) The interactions between R393 and aɛFab residues. (e) Stereo image of 2Fo–Fc electron density at 1σ contour level for residues around R393 (shown in orange in the center of the image) at the interface between IgE-FcB (orange) and aɛFab2 (green).

Supplementary Figure 3 Representation of the collective motions used as collective variables in the metadynamics simulation of Figure 2.

(a) Black arrow indicates collective motion 1 (x-axis in Figure 2), with the middle structure representing x=0. (b) Black arrow indicates collective motion 2 (y-axis in Figure 2) with the two structures representing the extremes explored across x=0. This motion is principally a twisting of (Cɛ2)2 relative to the Cɛ3-Cɛ4 domains. (c) Free-energy surface representing the IgE-Fc unbending process generated through metadynamics simulation. Axes show the projection along the two lowest frequency collective motions (extracted from a biased trajectory, as for Fig. 2a). This plot shows the features of the surface within 40 kJ/mol of the lowest free-energy minimum, contoured every 2.5 kJ/mol and coloured accordingly. (d) Short unbiased simulation starting from the extended conformation of IgE-Fc (Fig. 1c) in the crystal structure of the complex. The simulation was run for 250 ns and the trajectory is represented by a black line plotted over the free-energy surface (Fig. 2a). The simulation started at x = –0.4, y = 7.6 (indicated with black cross) and was terminated at x = 3.1, y = –7.3 (black circle). This trajectory is consistent with the small energy barriers surrounding the extended conformation.

Supplementary Figure 4 Stopped-flow kinetic analysis of aɛFab binding to IgE-Fc.

Kinetic binding curves showing the change in fluorescence when (a) aɛFab binds to IgE-Fc and (b) aɛFab binds to Fcɛ3-4. The red traces indicate experiments carried out with IgE-Fc or Fcɛ3-4 in excess over aɛFab, and the black traces are experiments with aɛFab in excess over IgE-Fc or Fcɛ3-4. The kinetic binding parameters demonstrate a linear concentration dependence for aɛFab binding to IgE-Fc (c and d) and Fcɛ3-4 (e and f).

Supplementary Figure 5 SPR analysis of aɛFab1–IgE-Fc and aɛFab1–IgE-Fc–aɛFab2 complex formation, and inhibition of sFcɛRIα binding by aɛFab1.

(a) SPR sensorgrams of IgE-Fc binding to immobilized aɛFab. IgE-Fc was injected over the surface at concentrations of 78 (orange), 156 (green), 313 (purple), 625 (magenta), 1250 (blue), 2500 (red), and 5000 nM (black). Data are fit to an equilibrium model of single site binding (inset). (b) aɛFab binds to pre-bound aɛFab1–IgE-Fc complex to form aɛFab1–IgE-Fc–aɛFab2. aɛFab was injected over the aɛFab1–IgE-Fc surface at concentrations of 0 (purple), 78 (orange), 156 (green), 313 (purple), 625 (magenta), 1250 (blue), 2500 (red), and 5000 nM (black). Inset shows aɛFab2 binding normalised with respect to the buffer only control (purple). (c) When IgE-Fc is bound to aɛFab1 on the SPR surface, sFcɛRIα is not able to bind. Inset shows sFcɛRIα binding normalised with respect to buffer only control. sFcɛRIα was injected over the aɛFab1–IgE-Fc surface at concentrations of 0 (orange), 31.3 (green), 62.5 (purple), 125 (magenta), 250 (blue), 500 (red), and 1000 nM (black).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 (PDF 2609 kb)

The structure of IgE-Fc bound symmetrically by two aɛFab molecules.

IgE-FcA is shown in blue and IgE-FcB in orange; aɛFab heavy chains are shown in green and light chains in grey. Also shown is the extended conformation of IgE-Fc as seen in the complex, which undergoes an “unbending” of 120° compared to the free structure. The unbending derives largely from hinge movement in the Cɛ2-Cɛ3 linker region (residues P333, R334, G335) as shown. (MOV 5434 kb)

The existence of an unbent conformation of IgE-Fc in the aɛFab complex suggests that the molecule may pass through an extended state as it flips between the two bent conformations.

This potential motion is shown in two orthogonal orientations, with IgE-FcA in blue and IgE-FcB in orange. (MOV 12543 kb)

Proposed mechanism of IgE-Fc flexibility and aɛFab binding in solution.

IgE-Fc is predominantly bent in solution, but (Cɛ2)2 may be capable of flipping from one side of the molecule to the other. aɛFab1 engages at either exposed binding site of IgE-Fc, attaching to the Cɛ3 domain, and limiting the range of accessible conformations. aɛFab2 engages while IgE-Fc transiently occupies the extended conformation, capturing the molecule in a symmetrical state. (MOV 3089 kb)

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Drinkwater, N., Cossins, B., Keeble, A. et al. Human immunoglobulin E flexes between acutely bent and extended conformations. Nat Struct Mol Biol 21, 397–404 (2014). https://doi.org/10.1038/nsmb.2795

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