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

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 options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Structure of the insulin receptor dimer.
Figure 2: Class 1 and class 2 dimers.
Figure 3: Proposed transduction mechanism.

Accession codes

Primary accessions

Electron Microscopy Data Bank

Protein Data Bank

References

  1. 1

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

  2. 2

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

  3. 3

    Frasca, F. 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

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

  5. 5

    Seino, S., Seino, M., Nishi, S. & Bell, G. I. Structure of the human insulin receptor gene and characterization of its promoter. Proc. Natl Acad. Sci. USA 86, 114–118 (1989)

  6. 6

    De Meyts, P. 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

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

  8. 8

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

  9. 9

    Smith, B. J. 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

    Croll, T. I. 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

    Whittaker, L., Hao, C., Fu, W. & Whittaker, J. High-affinity insulin binding: insulin interacts with two receptor ligand binding sites. Biochemistry 47, 12900–12909 (2008)

  12. 12

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

  13. 13

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

  14. 14

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

  15. 15

    Jain, T., Sheehan, P., Crum, J., Carragher, B. & Potter, C. S. Spotiton: a prototype for an integrated inkjet dispense and vitrification system for cryo-TEM. J. Struct. Biol. 179, 68–75 (2012)

  16. 16

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

  17. 17

    Markussen, J., Halstrøm, J., Wiberg, F. C. & Schäffer, L. Immobilized insulin for high capacity affinity chromatography of insulin receptors. J. Biol. Chem. 266, 18814–18818 (1991)

  18. 18

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

  19. 19

    Ward, C. W., Menting, J. G. & Lawrence, M. C. 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

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

  21. 21

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

  22. 22

    Accili, D., Mosthaf, L., Ullrich, A. & Taylor, S. I. 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

    Lebrun, C. 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

    Kadowaki, H. 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

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

  26. 26

    Ye, L. 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

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

  28. 28

    Schäffer, L. & Ljungqvist, L. 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

    Sparrow, L. G. 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

    Gutmann, T., Kim, K. H., Grzybek, M., Walz, T. & Coskun, U. Visualization of ligand-induced transmembrane signalling in the full-length human insulin receptor. J. Cell Biol. https://doi.org/10.1083/jcb.201711047 (2018)

  31. 31

    Subramanian, K., Fee, C. J., Fredericks, R., Stubbs, R. S. & Hayes, M. T. Insulin receptor–insulin interaction kinetics using multiplex surface plasmon resonance. J. Mol. Recognit. 26, 643–652 (2013)

  32. 32

    Johnson, J. D., Wong, M. L. & Rutter, W. J. Properties of the insulin receptor ectodomain. Proc. Natl Acad. Sci. USA 85, 7516–7520 (1988)

  33. 33

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

  34. 34

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

  35. 35

    Dandey, V. P. et al. Spotiton: new features and applications. J. Struct. Biol. https://doi.org/10.1016/j.jsb.2018.01.002 (2018)

  36. 36

    Wei, H. et al. Optimizing “self-wicking” nanowire grids. J. Struct. Biol. https://doi.org/10.1016/j.jsb.2018.01.001 (2018)

  37. 37

    Russo, C. J. & Passmore, L. A. Electron microscopy: ultrastable gold substrates for electron cryomicroscopy. Science 346, 1377–1380 (2014)

  38. 38

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

  39. 39

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

  40. 40

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

  41. 41

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

  42. 42

    Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017)

  43. 43

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

  44. 44

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

  45. 45

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

  46. 46

    Afonine, P., Headd, J., Terwilliger, T. & Adams, P. New tool: phenix.real_space_refine. Comput. Crystallogr. Newsl. 4, 43–44 (2013)

  47. 47

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

  48. 48

    Menting, J. G. 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

Affiliations

Authors

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.

Corresponding author

Correspondence to Giovanna Scapin.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Additional information

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 figures and tables

Extended Data Figure 1 Data collection and processing.

a, Representative micrograph of the insulin receptor–insulin complex. Images were collected on a Krios (Thermo Fisher) equipped with an energy filter and a Gatan K2 Counting camera; the magnification was set to 105,000×, with a calibrated pixel size of 1.10 Å. b, Representative 2D class averages as calculated with Cryosparc42. c, Schematic of data processing.

Extended Data Figure 2 Map of class 1 reconstructed using C2 symmetry to a resolution of 4.3 Å.

a, Gold standard FSC curve from Cryosparc42. b, Euler angle orientation distribution from Cryosparc42. c, Gold standard FSC curve as calculated in RELION43. d, Plot of the global half-map FSC (solid red line) and spread of directional resolution values (±1σ from mean, green dotted lines; the blue bars are a histogram of 100 such values evenly sampled over the 3D FSC14). e, Local resolution distribution (as calculated in Cryosparc42). f, Selected areas from the class 1 C2 map at a resolution of 4.3 Å (map contoured at 12σ). Left, individual β-strands in the L2 region are well separated; middle, bulky side chains are visible in the electron density; right, density for the L1 β-barrel.

Extended Data Figure 3 Map of class 1 reconstructed using C1 symmetry to a resolution of 4.7 Å.

a, Gold standard FSC curve from Cryosparc42. b, Euler angle orientation distribution from Cryosparc42. c, Gold standard FSC curve as calculated in RELION43. d, Plot of the global half-map FSC (solid red line) and spread of directional resolution values (±1σ from mean, green dotted lines; the blue bars are a histogram of 100 such values evenly sampled over the 3D FSC14). e, Local resolution distribution (as calculated in Cryosparc42).

Extended Data Figure 4 Insulin binding and crystallographic S2 site.

a, Map of the class 1 structure obtained without applying C2 symmetry, with the insulin receptor model shown as a cartoon. The map is asymmetric and only one of the FnIII-2 subdomains is clearly visible. b, Positioning of the FnIII-2 subdomain enables analysis of the relative position of the bound insulin (blue) and the proposed S2 site (shown as spheres, residues selected according to ref. 26: the insulin is between 55 and 62 Å away, and on the opposite side of the proposed S2). c, Overlay of free insulin (PDB ID: 1ZNI) onto the insulin bound to the insulin receptor.

Extended Data Figure 5 Insulin receptor sample characterization.

a, Coomassie-stained SDS–PAGE gel. The protein was solubilized in PBS at pH 7.2 and run on a 4–12% Bis–Tris gel in 1× MOPS. Molecular weight markers are labelled. Bands corresponding to the α- and β-chains are indicated. This experiment was only run once to confirm sample quality was as reported by R&D Systems. For gel source data, see Supplementary Fig. 1. b, Cryo-EM density for the un-symmetrized map (4.7 Å resolution) countered at 6σ. Density that can be attributed to the second FnIII-2 domain (blue arrow) as well as one of the FnIII-3 domains (red arrow) is visible, but it is not of sufficient quality for building the model.

Extended Data Figure 6 Map of class 2 reconstructed using C1 symmetry to a resolution of 7.4 Å.

a, Gold standard FSC curve from Cryosparc42. b, Euler angle orientation distribution from Cryosparc42. c, Gold standard FSC curve as calculated in RELION43. d, Plot of the global half-map FSC (solid red line) and spread of directional resolution values (±1σ from mean, green dotted lines; the blue bars are a histogram of 100 such values evenly sampled over the 3D FSC14). e, Local resolution distribution (as calculated in Cryosparc42).

Extended Data Figure 7 Comparison between the crystallographic and the cryo-EM dimer.

a, The top panel shows three different views of the cryo-EM dimer, related by the 90° rotation shown in the figure. These are surface (for one monomer) and cartoon (for the other) representations of the coordinates fitted to the un-symmetrized, 4.7 Å map. In this model one of the FnIII-2 subdomains is visible. Both monomers are coloured according to the subdomains (L1, blue; CR, red; L2, yellow; FnIII-1, green; FnIII-2, light blue). The bottom panel shows three corresponding views for the crystallographic dimer. One monomer is shown as surface, the other as a cartoon; both are coloured according to the subdomains (in the cryo-EM dimer, the additional FnIII-3 is coloured in light purple). The relative arrangements of the two monomers in the cryo-EM and crystallographic dimers are completely different. b, If the crystallographic insulin receptor dimer (left panel) represents the biological apo insulin receptor dimer, the transition to the insulin-bound dimer (right panel) would imply a putative intermediate state, shown in the middle panel. In this state, the large conformational changes required to accommodate the α-CT helix (orange ribbon) and insulin (white ribbon), and to allow them to engage the S2 site (black spheres) would require disruption of the extensive surface interface between L1 and FnIII-2′ and 3′ and L2-FnIII-1′ interactions. In the middle and right panels the α-CT helix and the insulin are from the cryo-EM structure.

Extended Data Figure 8 Comparison between the bound and unbound insulin receptor monomer.

Transition from the apo (magenta, PDB ID: 4ZXB monomer) to the bound form (green, cryo-EM structure) for the insulin receptor monomer. This transition can be described as two rotations with respect to the linker regions identified by the black arrows. The α-CT helix is shown as a cartoon.

Extended Data Table 1 Summary of the interactions between the insulin receptor and bound insulin
Extended Data Table 2 Human insulin receptor ECD binding and kinetic data

Supplementary information

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. (PDF 368 kb)

Life Sciences Reporting Summary (PDF 74 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Scapin, G., Dandey, V., Zhang, Z. et al. Structure of the insulin receptor–insulin complex by single-particle cryo-EM analysis. Nature 556, 122–125 (2018). https://doi.org/10.1038/nature26153

Download citation

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.