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Accelerated disassembly of IgE–receptor complexes by a disruptive macromolecular inhibitor

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Abstract

IgE antibodies bind the high-affinity IgE Fc receptor (FcεRI), found primarily on mast cells and basophils, and trigger inflammatory cascades of the allergic response1,2. Inhibitors of IgE–FcεRI binding have been identified and an anti-IgE therapeutic antibody (omalizumab) is used to treat severe allergic asthma3,4. However, preformed IgE–FcεRI complexes that prime cells before allergen exposure dissociate extremely slowly5 and cannot be disrupted by strictly competitive inhibitors. IgE-Fc conformational flexibility indicated that inhibition could be mediated by allosteric or other non-classical mechanisms6,7,8. Here we demonstrate that an engineered protein inhibitor, DARPin E2_79 (refs 9, 10, 11), acts through a non-classical inhibition mechanism, not only blocking IgE–FcεRI interactions, but actively stimulating the dissociation of preformed ligand–receptor complexes. The structure of the E2_79–IgE-Fc3-4 complex predicts the presence of two non-equivalent E2_79 sites in the asymmetric IgE–FcεRI complex, with site 1 distant from the receptor and site 2 exhibiting partial steric overlap. Although the structure is indicative of an allosteric inhibition mechanism, mutational studies and quantitative kinetic modelling indicate that E2_79 acts through a facilitated dissociation mechanism at site 2 alone. These results demonstrate that high-affinity IgE–FcεRI complexes can be actively dissociated to block the allergic response and suggest that protein–protein complexes may be more generally amenable to active disruption by macromolecular inhibitors.

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Figure 1: A fluorescence-quenching assay reveals different classes of IgE-directed inhibitors.
Figure 2: DARPin E2_79 binds IgE-Cε3 domains outside the FcεRIα binding site.
Figure 3: Binding and inhibition activities of E2_79 mutants.
Figure 4: Kinetic modelling of SPR data points to a facilitated dissociation mechanism for E2_79.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The structure factors and model for the E2_79–C335 IgE complex have been deposited in the Protein Data Bank under accession code 4GRG.

Change history

  • 22 November 2012

    This article was originally incorrectly listed under Planetary Sciences. The subject terms have since been corrected.

References

  1. Kraft, S. & Kinet, J. P. New developments in FcεRI regulation, function and inhibition. Nature Rev. Immunol. 7, 365–378 (2007)

    CAS  Article  Google Scholar 

  2. Gould, H. J. & Sutton, B. J. IgE in allergy and asthma today. Nature Rev. Immunol. 8, 205–217 (2008)

    CAS  Article  Google Scholar 

  3. D’Amato, G., Bucchioni, E., Oldani, V. & Canonica, W. Treating moderate-to-severe allergic asthma with a recombinant humanized anti-IgE monoclonal antibody (Omalizumab). Treat. Respir. Med. 5, 393–398 (2006)

    Article  Google Scholar 

  4. Chang, T. W. The pharmacological basis of anti-IgE therapy. Nature Biotechnol. 18, 157–162 (2000)

    CAS  Article  Google Scholar 

  5. Holdom, M. D. et al. Conformational changes in IgE contribute to its uniquely slow dissociation rate from receptor FcεRI. Nature Struct. Mol. Biol. 18, 571–576 (2011)

    CAS  Article  Google Scholar 

  6. Dhaliwal, B. et al. Crystal structure of IgE bound to its B-cell receptor CD23 reveals a mechanism of reciprocal allosteric inhibition with high affinity receptor FcεRI. Proc. Natl Acad. Sci. USA 109, 12686–12691 (2012)

    ADS  CAS  Article  Google Scholar 

  7. Wurzburg, B. A. & Jardetzky, T. S. Conformational flexibility in immunoglobulin E-Fc3–4 revealed in multiple crystal forms. J. Mol. Biol. 393, 176–190 (2009)

    CAS  Article  Google Scholar 

  8. Wurzburg, B. A., Garman, S. C. & Jardetzky, T. S. Structure of the human IgE-Fc Cε3-Cε4 reveals conformational flexibility in the antibody effector domains. Immunity 13, 375–385 (2000)

    CAS  Article  Google Scholar 

  9. Eggel, A., Baumann, M. J., Amstutz, P., Stadler, B. M. & Vogel, M. DARPins as bispecific receptor antagonists analyzed for immunoglobulin E receptor blockage. J. Mol. Biol. 393, 598–607 (2009)

    CAS  Article  Google Scholar 

  10. Baumann, M. J., Eggel, A., Amstutz, P., Stadler, B. M. & Vogel, M. DARPins against a functional IgE epitope. Immunol. Lett. 133, 78–84 (2010)

    CAS  Article  Google Scholar 

  11. Eggel, A. et al. Inhibition of ongoing allergic reactions using a novel anti-IgE DARPin-Fc fusion protein. Allergy 66, 961–968 (2011)

    CAS  Article  Google Scholar 

  12. Garman, S. C., Wurzburg, B. A., Tarchevskaya, S. S., Kinet, J. P. & Jardetzky, T. S. Structure of the Fc fragment of human IgE bound to its high-affinity receptor FcεRIα. Nature 406, 259–266 (2000)

    ADS  CAS  Article  Google Scholar 

  13. Basu, M. et al. Purification and characterization of human recombinant IgE-Fc fragments that bind to the human high affinity IgE receptor. J. Biol. Chem. 268, 13118–13127 (1993)

    CAS  PubMed  Google Scholar 

  14. Wang, B. et al. Epidermal Langerhans cells from normal human skin bind monomeric IgE via FcεRI. J. Exp. Med. 175, 1353–1365 (1992)

    CAS  Article  Google Scholar 

  15. Mirkina, I., Schweighoffer, T. & Kricek, F. Inhibition of human cord blood-derived mast cell responses by anti-FcεRI mAb 15/1 versus anti-IgE Omalizumab. Immunol. Lett. 109, 120–128 (2007)

    CAS  Article  Google Scholar 

  16. Mendonsa, S. D. & Bowser, M. T. In vitro selection of high-affinity DNA ligands for human IgE using capillary electrophoresis. Anal. Chem. 76, 5387–5392 (2004)

    CAS  Article  Google Scholar 

  17. Wiegand, T. W. et al. High-affinity oligonucleotide ligands to human IgE inhibit binding to Fcε receptor I. J. Immunol. 157, 221–230 (1996)

    CAS  PubMed  Google Scholar 

  18. Zheng, L. et al. Fine epitope mapping of humanized anti-IgE monoclonal antibody omalizumab. Biochem. Biophys. Res. Commun. 375, 619–622 (2008)

    CAS  Article  Google Scholar 

  19. Wright, J. D. & Lim, C. Prediction of an anti-IgE binding site on IgE. Protein Eng. 11, 421–427 (1998)

    CAS  Article  Google Scholar 

  20. Wurzburg, B. A. et al. An engineered disulfide bond reversibly traps the IgE-Fc3-4 in a closed, non-receptor binding conformation. J. Biol. Chem. http://dx.doi.org/10.1074/jbc.M112.407502 (2012)

  21. Prinz, H. & Striessnig, J. Ligand-induced accelerated dissociation of (+)-cis-diltiazem from L-type Ca2+ channels is simply explained by competition for individual attachment points. J. Biol. Chem. 268, 18580–18585 (1993)

    CAS  PubMed  Google Scholar 

  22. Gutfreund, H. Kinetics for the Life Sciences. Receptors, Transmitters and Catalysts (Cambridge Univ. Press, 1995)

    Book  Google Scholar 

  23. Hoops, S. et al. COPASI—a COmplex PAthway SImulator. Bioinformatics 22, 3067–3074 (2006)

    CAS  Article  Google Scholar 

  24. He, M. M. et al. Small-molecule inhibition of TNF-α. Science 310, 1022–1025 (2005)

    ADS  CAS  Article  Google Scholar 

  25. Wells, J. A. & McClendon, C. L. Reaching for high-hanging fruit in drug discovery at protein-protein interfaces. Nature 450, 1001–1009 (2007)

    ADS  CAS  Article  Google Scholar 

  26. Wang, C. C. et al. Negative and positive site-site interactions, and their modulation by pH, insulin analogs, and monoclonal antibodies, are preserved in the purified insulin receptor. Proc. Natl Acad. Sci. USA 85, 8400–8404 (1988)

    ADS  CAS  Article  Google Scholar 

  27. Lowenthal, J. W. et al. High and low affinity IL 2 receptors: analysis by IL 2 dissociation rate and reactivity with monoclonal anti-receptor antibody PC61. J. Immunol. 135, 3988–3994 (1985)

    CAS  PubMed  Google Scholar 

  28. Boulain, J. C. & Menez, A. Neurotoxin-specific immunoglobulins accelerate dissociation of the neurotoxin-acetylcholine receptor complex. Science 217, 732–733 (1982)

    ADS  CAS  Article  Google Scholar 

  29. Garman, S. C., Kinet, J. P. & Jardetzky, T. S. Crystal structure of the human high-affinity IgE receptor. Cell 95, 951–961 (1998)

    CAS  Article  Google Scholar 

  30. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. in Methods Enzymol. Vol. 276 (eds Carter, J. C. W. & Sweet, R. M. ) 307–326 (Academic, 1997)

    Google Scholar 

  31. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)

    CAS  Article  Google Scholar 

  32. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

    Google Scholar 

  33. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010)

    CAS  Article  Google Scholar 

  34. Laskowski, R. A., Rullmannn, J. A., MacArthur, M. W., Kaptein, R. & Thornton, J. M. AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J. Biomol. NMR 8, 477–486 (1996)

    CAS  Article  Google Scholar 

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Acknowledgements

We thank B. Wurzburg and other past and present members of the Jardetzky Laboratory. This research was supported in part by an NIH research grant (AI-18939) and an American Asthma Foundation Senior Investigator Award as well as the Swiss National Science Foundation grant number 310030_127350 to T.S.J. We also thank C. A. Dahinden and B. M. Stadler and members of their groups for valuable discussions. Furthermore, we thank M. J. Baumann for technical support and Molecular Partners AG, especially P. Amstutz, M. T. Stumpp, P. Forrer and D. Steiner, for placing DARPin libraries at our disposal and providing scientific support.

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Contributions

B.K., A.E., S.S.T. and T.S.J. designed and performed experiments. B.K., A.E., S.S.T., H.P. and T.S.J. analysed data. B.K., S.S.T., A.E., M.V. and T.S.J. contributed reagents. B.K., S.S.T., A.E., M.V., H.P. and T.S.J. discussed/commented on results and edited the manuscript. B.K., A.E. and T.S.J. wrote the manuscript and Supplementary Information, and prepared the figures.

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Correspondence to Theodore S. Jardetzky.

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The authors declare no competing financial interests.

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Kim, B., Eggel, A., Tarchevskaya, S. et al. Accelerated disassembly of IgE–receptor complexes by a disruptive macromolecular inhibitor. Nature 491, 613–617 (2012). https://doi.org/10.1038/nature11546

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