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How insulin engages its primary binding site on the insulin receptor

Nature volume 493, pages 241–245 (2013)Cite this article

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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.

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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

Author notes

  1. John G. Menting and Jonathan Whittaker: These authors contributed equally to this work.

  2. Guy G. Dodson: Deceased.

Authors and Affiliations

  1. Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3052, Australia,

    John G. Menting, Mai B. Margetts, Geoffrey K.-W. Kong, Brian J. Smith, Colin W. Ward & Michael C. Lawrence

  2. Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, USA,

    Jonathan Whittaker, Linda J. Whittaker & Michael A. Weiss

  3. Department of Chemistry, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia,

    Brian J. Smith

  4. Department of Chemistry, York Structural Biology Laboratory, University of York, York YO10 5DD, UK

    Christopher J. Watson, Guy G. Dodson & Andrzej M. Brzozowski

  5. Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i., 16610 Prague, Czech Republic,

    Lenka Žáková, Emília Kletvíková & Jiří Jiráček

  6. Department of Medicine, University of Chicago, Chicago, Illinois 60637, USA,

    Shu Jin Chan & Donald F. Steiner

  7. Department of Medical Biology, University of Melbourne, Royal Parade, Parkville, Victoria 3010, Australia,

    Michael C. Lawrence

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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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