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How insulin engages its primary binding site on the insulin receptor

Abstract

Insulin receptor signalling has a central role in mammalian biology, regulating cellular metabolism, growth, division, differentiation and survival1,2. Insulin resistance contributes to the pathogenesis of type 2 diabetes mellitus and the onset of Alzheimer’s disease3; aberrant signalling occurs in diverse cancers, exacerbated by cross-talk with the homologous type 1 insulin-like growth factor receptor (IGF1R)4. Despite more than three decades of investigation, the three-dimensional structure of the insulin–insulin receptor complex has proved elusive, confounded by the complexity of producing the receptor protein. Here we present the first view, to our knowledge, of the interaction of insulin with its primary binding site on the insulin receptor, on the basis of four crystal structures of insulin bound to truncated insulin receptor constructs. The direct interaction of insulin with the first leucine-rich-repeat domain (L1) of insulin receptor is seen to be sparse, the hormone instead engaging the insulin receptor carboxy-terminal α-chain (αCT) segment, which is itself remodelled on the face of L1 upon insulin binding. Contact between insulin and L1 is restricted to insulin B-chain residues. The αCT segment displaces the B-chain C-terminal β-strand away from the hormone core, revealing the mechanism of a long-proposed conformational switch in insulin upon receptor engagement. This mode of hormone–receptor recognition is novel within the broader family of receptor tyrosine kinases5. We support these findings by photo-crosslinking data that place the suggested interactions into the context of the holoreceptor and by isothermal titration calorimetry data that dissect the hormone–insulin receptor interface. Together, our findings provide an explanation for a wealth of biochemical data from the insulin receptor and IGF1R systems relevant to the design of therapeutic insulin analogues.

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Figure 1: Structure of insulin, insulin receptor and the site 1 complexes.
Figure 2: The insulin–site 1 interaction.
Figure 3: Insulin interactions in L1–CR–L2 mini-insulin receptor and holo -insulin receptor.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomiccoordinates andstructure factors for complexes A, B,Cand D have been deposited with the Protein Data Bank under accession codes 3W11, 3W12, 3W13 and 3W14, respectively.

References

  1. Taniguchi, C. M., Emanuelli, B. & Kahn, C. R. Critical nodes in signalling pathways: insights into insulin action. Nature Rev. Mol. Cell Biol. 7, 85–96 (2006)

    Article  CAS  Google Scholar 

  2. Cohen, P. The twentieth century struggle to decipher insulin signalling. Nature Rev. Mol. Cell Biol. 7, 867–873 (2006)

    Article  CAS  Google Scholar 

  3. Talbot, K. et al. Demonstrated brain insulin resistance in Alzheimer’s disease patients is associated with IGF-1 resistance, IRS-1 dysregulation, and cognitive decline. J. Clin. Invest. 122, 1316–1338 (2012)

    Article  CAS  Google Scholar 

  4. Pollak, M. The insulin and insulin-like growth factor receptor family in neoplasia: an update. Nature Rev. Cancer 12, 159–169 (2012)

    Article  CAS  Google Scholar 

  5. Lemmon, M. A. & Schlessinger, J. Cell signaling by receptor tyrosine kinases. Cell 141, 1117–1134 (2010)

    Article  CAS  Google Scholar 

  6. Adams, M. J. et al. Structure of rhombohedral 2 zinc insulin crystals. Nature 224, 491–495 (1969)

    Article  ADS  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  8. Seino, S. & Bell, G. I. Alternative splicing of human insulin receptor messenger RNA. Biochem. Biophys. Res. Commun. 159, 312–316 (1989)

    Article  CAS  Google Scholar 

  9. Schäffer, L. A model for insulin binding to the insulin receptor. Eur. J. Biochem. 221, 1127–1132 (1994)

    Article  Google Scholar 

  10. De Meyts, P. Insulin and its receptor: structure, function and evolution. Bioessays 26, 1351–1362 (2004)

    Article  CAS  Google Scholar 

  11. Ward, C. W. & Lawrence, M. C. Ligand-induced activation of the insulin receptor: a multi-step process involving structural changes in both the ligand and the receptor. Bioessays 31, 422–434 (2009)

    Article  CAS  Google Scholar 

  12. 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)

    Article  ADS  CAS  Google Scholar 

  13. Ward, C. W. & Lawrence, M. C. Similar but different: ligand-induced activation of the insulin and epidermal growth factor receptor families. Curr. Opin. Struct. Biol. 22, 360–366 (2012)

    Article  CAS  Google Scholar 

  14. Kiselyov, V. V., Versteyhe, S., Gauguin, L. & De Meyts, P. Harmonic oscillator model of the insulin and IGF1 receptors’ allosteric binding and activation. Mol. Syst. Biol. 5, 243 (2009)

    Article  Google Scholar 

  15. 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)

    Article  CAS  Google Scholar 

  16. Lou, M. et al. The first three domains of the insulin receptor differ structurally from the insulin-like growth factor 1 receptor in the regions governing ligand specificity. Proc. Natl Acad. Sci. USA 103, 12429–12434 (2006)

    Article  ADS  CAS  Google Scholar 

  17. Garrett, T. P. et al. Crystal structure of the first three domains of the type-1 insulin-like growth factor receptor. Nature 394, 395–399 (1998)

    Article  ADS  CAS  Google Scholar 

  18. Jiráček, J. et al. Implications for the active form of human insulin based on the structural convergence of highly active hormone analogues. Proc. Natl Acad. Sci. USA 107, 1966–1970 (2010)

    Article  ADS  Google Scholar 

  19. Karplus, P. A. & Diederichs, K. Linking crystallographic model and data quality. Science 336, 1030–1033 (2012)

    Article  ADS  CAS  Google Scholar 

  20. Evans, P. Resolving some old problems in protein crystallography. Science 336, 986–987 (2012)

    Article  ADS  CAS  Google Scholar 

  21. Brünger, A. T., DeLaBarre, B., Davies, J. M. & Weis, W. I. X-ray structure determination at low resolution. Acta Crystallogr. D 65, 128–133 (2009)

    Article  Google Scholar 

  22. Smart, O. S. et al. Exploiting structure similarity in refinement: automated NCS and target-structure restraints in BUSTER. Acta Crystallogr. D 68, 368–380 (2012)

    Article  CAS  Google Scholar 

  23. Murshudov, G. N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D 67, 355–367 (2011)

    Article  CAS  Google Scholar 

  24. Hua, Q. X., Shoelson, S. E., Kochoyan, M. & Weiss, M. A. Receptor binding redefined by a structural switch in a mutant human insulin. Nature 354, 238–241 (1991)

    Article  ADS  CAS  Google Scholar 

  25. Whittaker, J. et al. α-Helical element at the hormone-binding surface of the insulin receptor functions as a signaling element to activate its tyrosine kinase. Proc. Natl Acad. Sci. USA 109, 11166–11171 (2012)

    Article  ADS  CAS  Google Scholar 

  26. Kristensen, C., Wiberg, F. C. & Andersen, A. S. Specificity of insulin and insulin-like growth factor I receptors investigated using chimeric mini-receptors. Role of C-terminal of receptor α subunit. J. Biol. Chem. 274, 37351–37356 (1999)

    Article  CAS  Google Scholar 

  27. Xu, B. et al. Diabetes-associated mutations in insulin: consecutive residues in the B chain contact distinct domains of the insulin receptor. Biochemistry 43, 8356–8372 (2004)

    Article  CAS  Google Scholar 

  28. Xu, B. et al. Decoding the cryptic active conformation of a protein by synthetic photo-scanning. Insulin inserts a detachable arm between receptor domains. J. Biol. Chem. 284, 14597–14608 (2009)

    Article  CAS  Google Scholar 

  29. Xu, B. et al. Diabetes-associated mutations in insulin identify invariant receptor contacts. Diabetes 53, 1599–1602 (2004)

    Article  CAS  Google Scholar 

  30. Weiss, M. S. Global indicators of X-ray data quality. J. Appl. Cryst. 34, 130–135 (2001)

    Article  CAS  Google Scholar 

  31. Hoogenboom, H. R. et al. Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. Nucleic Acids Res. 19, 4133–4137 (1991)

    Article  CAS  Google Scholar 

  32. Stanley, P. Chinese hamster ovary cell mutants with multiple glycosylation defects for production of glycoproteins with minimal carbohydrate heterogeneity. Mol. Cell. Biol. 9, 377–383 (1989)

    Article  CAS  Google Scholar 

  33. Soos, M. A. et al. Monoclonal antibodies reacting with multiple epitopes on the human insulin receptor. Biochem. J. 235, 199–208 (1986)

    Article  CAS  Google Scholar 

  34. Evan, G. I., Lewis, G. K., Ramsay, G. & Bishop, J. M. Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol. Cell. Biol. 5, 3610–3616 (1985)

    Article  CAS  Google Scholar 

  35. Denley, A. et al. Structural determinants for high-affinity binding of insulin-like growth factor II to insulin receptor (IR)-A, the exon 11 minus isoform of the IR. Mol. Endocrinol. 18, 2502–2512 (2004)

    Article  CAS  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  37. Žáková, L. et al. Insulin analogues with modifications at position B26. Divergence of binding affinity and biological activity. Biochemistry 47, 5858–5868 (2008)

    Article  Google Scholar 

  38. McPhillips, T. M. et al. Blu-ice and the distributed control system: software for data acquisition and instrument control at macromolecular crystallography beamlines. J. Synchrotron Radiat. 9, 401–406 (2002)

    Article  CAS  Google Scholar 

  39. Kabsch, W. Integration, scaling, space-group assignment and post-refinement. Acta Crystallogr. D 66, 133–144 (2010)

    Article  CAS  Google Scholar 

  40. Collaborative Computing Project The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)

    Article  Google Scholar 

  41. McCoy, A. J. Solving structures of protein complexes by molecular replacement with Phaser. Acta Crystallogr. D 63, 32–41 (2007)

    Article  CAS  Google Scholar 

  42. Bricogne, G. et al. BUSTER version 2.10 (Global Phasing Ltd, 2011)

  43. Blanc, E. et al. Refinement of severely incomplete structures with maximum likelihood in BUSTER-TNT. Acta Crystallogr. D 60, 2210–2221 (2004)

    Article  CAS  Google Scholar 

  44. Kleywegt, G. J. & Jones, T. A. Template convolution to enhance or detect structural features in macromolecular electron-density maps. Acta Crystallogr. D 53, 179–185 (1997)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  47. Jones, T. A. & Thirup, S. Using known substructures in protein model building and crystallography. EMBO J. 5, 819–822 (1986)

    Article  CAS  Google Scholar 

  48. Lüthy, R., Bowie, J. U. & Eisenberg, D. Assessment of protein models with three-dimensional profiles. Nature 356, 83–85 (1992)

    Article  ADS  Google Scholar 

  49. Sparrow, L. G. et al. N-linked glycans of the human insulin receptor and their distribution over the crystal structure. Proteins Struct. Funct. Bioinform. 71, 426–439 (2008)

    Article  CAS  Google Scholar 

  50. Vagin, A. & Teplyakov, A. Molecular replacement with MOLREP. Acta Crystallogr. D 66, 22–25 (2010)

    Article  CAS  Google Scholar 

  51. Hua, Q. X. et al. Enhancing the activity of a protein by stereospecific unfolding. The conformational life cycle of insulin and its evolutionary origins. J. Biol. Chem. 248, 14586–14596 (2009)

    Article  Google Scholar 

  52. Menting, J. G., Ward, C. W., Margetts, M. B. & Lawrence, M. C. A thermodynamic study of ligand binding to the first three domains of the human insulin receptor: relationship between the receptor α-chain C-terminal peptide and the site 1 insulin mimetic peptides. Biochemistry 48, 5492–5500 (2009)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This Letter is dedicated to our co-author, the late Guy Dodson, in recognition of his lifetime contribution to the study of the structure of insulin. This work was supported by Australian National Health and Medical Research Council (NHMRC) Project grants 516729, 575539 and 1005896 and the Hazel and Pip Appel Fund (to M.C.L.), NHMRC Independent Research Institutes Infrastructure Support Scheme Grant 361646 and Victorian State Government Operational Infrastructure Support Grant (to the Walter and Eliza Hall Institute of Medical Research), NIH grant no. DK40949 (to M.A.W. and J.W.) and American Diabetes Association grant no. 1-11INI-31 (to J.W.), Grant Agency of the Czech Republic grant P207/11/P430 (to L.Z.), Research Project of the Academy of Sciences of the Czech Republic RVO:61388963 (to the Institute of Organic Chemistry and Biochemistry), NIH grants DK13914 and DK20595 (to D.F.S.), a BBSRC PhD studentship (to C.J.W.) and the UoY Research Priming Fund (to the York Structural Biology Laboratory). Part of this research was undertaken on the MX2 beamline at the Australian Synchrotron (AS), Victoria, Australia. We thank the DLS for access to beamline I24 and the Australian International Synchrotron Access Program for travel funds. We thank P. Colman and J. Gulbis, our colleagues at CSIRO and the AS beam line staff for their support; J. Turkenburg for assistance in collecting data at DLS; K. Huang for assistance with midi-receptor photo-crosslinking; Q.-X. Hua and Y. Yang for discussion of NMR studies of insulin; S.-Q. Hu, S. H. Nakagawa, N. F. Phillips and S. Wang for assistance with insulin analogue synthesis; P. G. Katsoyannis for advice about the synthesis of photo-reactive insulin analogues and for providing an initial set of Pap analogues; K. Siddle for supplying the 83-7 and 83-14 hybridomas; L. Lu and the fermentation group CSIRO Materials Science and Engineering for large-scale cell culture.

Author information

Authors and Affiliations

Authors

Contributions

J.G.M. and J.W. contributed equally to the paper. J.G.M. purified and crystallized samples, collected data and performed the ITC study; J.W. and L.J.W. performed receptor photo-crosslinking experiments; M.B.M. performed molecular biology, cell culture and crystallization experiments; S.J.C. performed insulin photo-crosslinking experiments; G.K.-W.K. and C.J.W. performed crystallography experiments; B.J.S. performed calculations; E.K., L.Z. and J.J. prepared insulin analogues; C.W.W., M.A.W., J.W., D.F.S., S.J.C., J.G.M. and M.C.L. designed the experiments and analysed data. C.W.W., M.A.W., A.M.B., G.G.D. and M.C.L. wrote the paper. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Michael A. Weiss or Michael C. Lawrence.

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Competing interests

J.W. is a consultant to Thermalin Diabetes, LLC; J.W. and L.J.W. own stock in Novo-Nordisk A/S; M.A.W. owns stock in Thermalin Diabetes, LLC, serves as its Chief Scientific Officer, and is a member of its Board of Directors, and has also served as a consultant to Merck and DEKA R&D Corporation. M.C.L. has received honoraria and/or travel funding for seminars delivered at Novo-Nordisk A/S and Sanofi-Aventis.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-10, Supplementary Tables 1-4, a Supplementary Discussion and Supplementary References. The Supplementary Information provides additional background information to the insulin receptor system, images showing how the Fabs bind to the receptor constructs, images showing the assembly of the Complex D tetramer, an image showing the comparison of the conformations of insulin observed here with that of its receptor-free form and an image showing the implications for Site 2 binding. In addition, it provides protein production, purification, isothermal calorimetric and crystallographic detail. (PDF 11239 kb)

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Menting, J., Whittaker, J., Margetts, M. et al. How insulin engages its primary binding site on the insulin receptor. Nature 493, 241–245 (2013). https://doi.org/10.1038/nature11781

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