Letter | Published:

Structure of the insulin receptor–insulin complex by single-particle cryo-EM analysis

Nature volume 556, pages 122125 (05 April 2018) | Download Citation

Abstract

The insulin receptor is a dimeric protein that has a crucial role in controlling glucose homeostasis, regulating lipid, protein and carbohydrate metabolism, and modulating brain neurotransmitter levels1,2. Insulin receptor dysfunction has been associated with many diseases, including diabetes, cancer and Alzheimer’s disease1,3,4. The primary sequence of the receptor has been known since the 1980s5, and is composed of an extracellular portion (the ectodomain, ECD), a single transmembrane helix and an intracellular tyrosine kinase domain. Binding of insulin to the dimeric ECD triggers auto-phosphorylation of the tyrosine kinase domain and subsequent activation of downstream signalling molecules. Biochemical and mutagenesis data have identified two putative insulin-binding sites, S1 and S26. The structures of insulin bound to an ECD fragment containing S1 and of the apo ectodomain have previously been reported7,8, but details of insulin binding to the full receptor and the signal propagation mechanism are still not understood. Here we report single-particle cryo-electron microscopy reconstructions of the 1:2 (4.3 Å) and 1:1 (7.4 Å) complexes of the insulin receptor ECD dimer with insulin. The symmetrical 4.3 Å structure shows two insulin molecules per dimer, each bound between the leucine-rich subdomain L1 of one monomer and the first fibronectin-like domain (FnIII-1) of the other monomer, and making extensive interactions with the α-subunit C-terminal helix (α-CT helix). The 7.4 Å structure has only one similarly bound insulin per receptor dimer. The structures confirm the binding interactions at S1 and define the full S2 binding site. These insulin receptor states suggest that recruitment of the α-CT helix upon binding of the first insulin changes the relative subdomain orientations and triggers downstream signal propagation.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Primary accessions

Electron Microscopy Data Bank

References

  1. 1.

    & Insulin signalling and the regulation of glucose and lipid metabolism. Nature 414, 799–806 (2001)

  2. 2.

    , & Insulin and insulin-like growth factor receptors in the nervous system. Mol. Neurobiol. 3, 71–100 (1989)

  3. 3.

    et al. The role of insulin receptors and IGF-I receptors in cancer and other diseases. Arch. Physiol. Biochem. 114, 23–37 (2008)

  4. 4.

    Alzheimer disease: insulin resistance and AD—extending the translational path. Nat. Rev. Neurol. 8, 360–362 (2012)

  5. 5.

    , , & Structure of the human insulin receptor gene and characterization of its promoter. Proc. Natl Acad. Sci. USA 86, 114–118 (1989)

  6. 6.

    Insulin/receptor binding: the last piece of the puzzle? What recent progress on the structure of the insulin/receptor complex tells us (or not) about negative cooperativity and activation. BioEssays 37, 389–397 (2015)

  7. 7.

    et al. How insulin engages its primary binding site on the insulin receptor. Nature 493, 241–245 (2013)

  8. 8.

    et al. Structure of the insulin receptor ectodomain reveals a folded-over conformation. Nature 443, 218–221 (2006)

  9. 9.

    et al. Structural resolution of a tandem hormone-binding element in the insulin receptor and its implications for design of peptide agonists. Proc. Natl Acad. Sci. USA 107, 6771–6776 (2010)

  10. 10.

    et al. Higher-resolution structure of the human insulin receptor ectodomain: multi-modal inclusion of the insert domain. Structure 24, 469–476 (2016)

  11. 11.

    , , & High-affinity insulin binding: insulin interacts with two receptor ligand binding sites. Biochemistry 47, 12900–12909 (2008)

  12. 12.

    Structural dynamics of insulin receptor and transmembarne signaling. Biochemistry 54, 5523–5532 (2015)

  13. 13.

    & Structural biology of insulin and IGF1 receptors: implications for drug design. Nat. Rev. Drug Discov. 1, 769–783 (2002)

  14. 14.

    et al. Addressing preferred specimen orientation in single-particle cryo-EM through tilting. Nat. Methods 14, 793–796 (2017)

  15. 15.

    , , , & Spotiton: a prototype for an integrated inkjet dispense and vitrification system for cryo-TEM. J. Struct. Biol. 179, 68–75 (2012)

  16. 16.

    et al. A new method for vitrifying samples for cryoEM. J. Struct. Biol. 195, 190–198 (2016)

  17. 17.

    , , & Immobilized insulin for high capacity affinity chromatography of insulin receptors. J. Biol. Chem. 266, 18814–18818 (1991)

  18. 18.

    & Characterization of the functional insulin binding epitopes of the full-length insulin receptor. J. Biol. Chem. 280, 20932–20936 (2005)

  19. 19.

    , & The insulin receptor changes conformation in unforeseen ways on ligand binding: sharpening the picture of insulin receptor activation. BioEssays 35, 945–954 (2013)

  20. 20.

    , & Characterization of a second ligand binding site of the insulin receptor. Biochem. Biophys. Res. Commun. 347, 334–339 (2006)

  21. 21.

    et al. A novel human insulin receptor gene mutation uniquely inhibits insulin binding without impairing posttranslational processing. Diabetes 43, 1096–1102 (1994)

  22. 22.

    , , & A mutation in the extracellular domain of the insulin receptor impairs the ability of insulin to stimulate receptor autophosphorylation. J. Biol. Chem. 266, 434–439 (1991)

  23. 23.

    et al. Antibodies to the extracellular receptor domain restore the hormone-insensitive kinase and conformation of the mutant insulin receptor valine 382. J. Biol. Chem. 268, 11272–11277 (1993)

  24. 24.

    et al. Mutagenesis of lysine 460 in the human insulin receptor. Effects upon receptor recycling and cooperative interactions among binding sites. J. Biol. Chem. 265, 21285–21296 (1990)

  25. 25.

    , & Dimeric fragment of the insulin receptor α-subunit binds insulin with full holoreceptor affinity. J. Biol. Chem. 276, 12378–12384 (2001)

  26. 26.

    et al. Structure and dynamics of the insulin receptor: implications for receptor activation and drug discovery. Drug Discov. Today 22, 1092–1102 (2017)

  27. 27.

    et al. An open-and-shut case? Recent insights into the activation of EGF/ErbB receptors. Mol. Cell 12, 541–552 (2003)

  28. 28.

    & Identification of a disulfide bridge connecting the α-subunits of the extracellular domain of the insulin receptor. Biochem. Biophys. Res. Commun. 189, 650–653 (1992)

  29. 29.

    et al. The disulfide bonds in the C-terminal domains of the human insulin receptor ectodomain. J. Biol. Chem. 272, 29460–29467 (1997)

  30. 30.

    , , , & Visualization of ligand-induced transmembrane signalling in the full-length human insulin receptor. J. Cell Biol. (2018)

  31. 31.

    , , , & Insulin receptor–insulin interaction kinetics using multiplex surface plasmon resonance. J. Mol. Recognit. 26, 643–652 (2013)

  32. 32.

    , & Properties of the insulin receptor ectodomain. Proc. Natl Acad. Sci. USA 85, 7516–7520 (1988)

  33. 33.

    et al. Purification and properties of insulin receptor ectodomain from large-scale mammalian cell culture. Protein Expr. Purif. 6, 789–798 (1995)

  34. 34.

    Human insulin from recombinant DNA technology. Science 219, 632–637 (1983)

  35. 35.

    et al. Spotiton: new features and applications. J. Struct. Biol. (2018)

  36. 36.

    et al. Optimizing “self-wicking” nanowire grids. J. Struct. Biol. (2018)

  37. 37.

    & Electron microscopy: ultrastable gold substrates for electron cryomicroscopy. Science 346, 1377–1380 (2014)

  38. 38.

    & Opinion: hazards faced by macromolecules when confined to thin aqueous films. Biophys. Rep. 3, 1–7 (2017)

  39. 39.

    et al. Automated molecular microscopy: the new Leginon system. J. Struct. Biol. 151, 41–60 (2005)

  40. 40.

    et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017)

  41. 41.

    Gctf: Real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016)

  42. 42.

    , , & A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017)

  43. 43.

    A Bayesian view on cryo-EM structure determination. J. Mol. Biol. 415, 406–418 (2012)

  44. 44.

    & MOLREP: an automated program for molecular replacement. J. Appl. Cryst. 30, 1022–1025 (1997)

  45. 45.

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

  46. 46.

    , , & New tool: phenix.real_space_refine. Comput. Crystallogr. Newsl. 4, 43–44 (2013)

  47. 47.

    et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004)

  48. 48.

    et al. Protective hinge in insulin opens to enable its receptor engagement. Proc. Natl Acad. Sci. USA 111, E3395–E3404 (2014)

Download references

Acknowledgements

The work presented here was conducted at the National Resource for Automated Molecular Microscopy located at the New York Structural Biology Center, supported by grants from the NIH (GM103310, OD019994) and the Simons Foundation (349247). The authors would like to acknowledge the entire staff of the Simons Electron Microscopy Center at the New York Structural Biology Center for continuous help and technical support and G. Boykow, A. Ogawa and L. Zhang (In Vitro Pharmacology Group, Merck & Co.) for providing assay support.

Author information

Author notes

    • Giovanna Scapin
    •  & Venkata P. Dandey

    These authors contributed equally to this work.

Affiliations

  1. Merck & Co., Department of Biochemical Engineering & Structure, 2000 Galloping Hill Road, Kenilworth, New Jersey 07033, USA

    • Giovanna Scapin
    • , Winifred Prosise
    • , Alan Hruza
    •  & Corey Strickland
  2. Simons Electron Microscopy Center, National Resource for Automated Molecular Microscopy, New York Structural Biology Center, 89 Convent Avenue, New York, New York 10027, USA

    • Venkata P. Dandey
    • , Zhening Zhang
    • , Clinton S. Potter
    •  & Bridget Carragher
  3. Merck & Co., Department of Biophysics, NMR & Protein Products Characterization, 2000 Galloping Hill Road, Kenilworth, New Jersey 07033, USA

    • Theresa Kelly
    •  & Todd Mayhood

Authors

  1. Search for Giovanna Scapin in:

  2. Search for Venkata P. Dandey in:

  3. Search for Zhening Zhang in:

  4. Search for Winifred Prosise in:

  5. Search for Alan Hruza in:

  6. Search for Theresa Kelly in:

  7. Search for Todd Mayhood in:

  8. Search for Corey Strickland in:

  9. Search for Clinton S. Potter in:

  10. Search for Bridget Carragher in:

Contributions

G.S. collected and processed the data, interpreted the results and wrote the manuscript. V.P.D. prepared the grids used in the studies, collected and processed the data and assisted in writing the manuscript. Z.Z. prepared the grids used in the study. W.P. and A.H. obtained and prepared the receptor and insulin samples. T.K. produced the binding data. T.M. characterized the sample. Z.Z., W.P., A.H., T.K. and T.M. helped in writing the manuscript. C.S., C.S.P. and B.C. helped in planning the experiments, analysing the data and writing and editing of the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Giovanna Scapin.

Reviewer Information Nature thanks J. Rubinstein, G. Skiniotis and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Figure 1

    This file contains the uncropped scan of the SDS gel used to characterize the IR sample. Molecular weight markers are reported to the left of the scan. The rectangular box indicates the portion of the scan reported in Extended Data Figure 5.

  2. 2.

    Life Sciences Reporting Summary

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature26153

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.