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Structure of the insulin receptor ectodomain reveals a folded-over conformation

Abstract

The insulin receptor is a phylogenetically ancient tyrosine kinase receptor found in organisms as primitive as cnidarians and insects. In higher organisms it is essential for glucose homeostasis1, whereas the closely related insulin-like growth factor receptor (IGF-1R) is involved in normal growth and development2. The insulin receptor is expressed in two isoforms, IR-A and IR-B; the former also functions as a high-affinity receptor for IGF-II and is implicated, along with IGF-1R, in malignant transformation3. Here we present the crystal structure at 3.8 Å resolution of the IR-A ectodomain dimer, complexed with four Fabs from the monoclonal antibodies 83-7 and 83-14 (ref. 4), grown in the presence of a fragment of an insulin mimetic peptide5. The structure reveals the domain arrangement in the disulphide-linked ectodomain dimer, showing that the insulin receptor adopts a folded-over conformation that places the ligand-binding regions in juxtaposition. This arrangement is very different from previous models6. It shows that the two L1 domains are on opposite sides of the dimer, too far apart to allow insulin to bind both L1 domains simultaneously as previously proposed7. Instead, the structure implicates the carboxy-terminal surface of the first fibronectin type III domain as the second binding site involved in high-affinity binding.

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Figure 1: The IRΔβ ectodomain homodimer, showing the juxtaposition of domains between the monomers.
Figure 2: The IRΔβ ectodomain homodimer, showing the attached pairs of Fabs.
Figure 3: Comparison of the crystal structure of the Fab-complexed IRΔβ ectodomain with single-particle electron microscope images.
Figure 4: Insulin bridging two monomers in the IRΔβ homodimer.

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Acknowledgements

We thank K. Siddle for the 83-7 and 83-14 monoclonal antibody cell lines; O. Dolezal for advice on cloning and sequencing the monoclonal antibody variable region cDNAs; B. Van Donkelaar for technical crystallography inputs; and L. Lu, L. Cheong and T. Phan for contributions to protein production. We acknowledge the help provided by beamline staff at both the Advanced Photon Source and the Photon Factory. This work was supported by the Australian Synchrotron Research Program, which is funded by the Commonwealth of Australia under the Major National Research Facilities Program. Additional financial support was provided under the Generic Technology component of the Industry Research and Development Act 1986, from Biota Diabetes Research Pty Ltd.

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Correspondence to Michael C. Lawrence or Colin W. Ward.

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Competing interests

Atomic coordinates of the IR-Fab complex have been deposited in the Protein Data Bank with accession number 2DTG. Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.

Supplementary information

Supplementary Notes

Models of insulin/IR binding. This file contains an overview of the models for insulin binding to the insulin receptor plus references. (DOC 31 kb)

Supplementary Methods

This file contains expanded methods details and references. (DOC 43 kb)

Supplementary Table 1

Diffraction data and refinement statistics. (DOC 35 kb)

Supplementary Figure 1

Schematic diagram of the insulin receptor dimer. (JPG 195 kb)

Supplementary Figure 2

Sequence alignment of the ectodomains of human insulin receptor (IR, exon 11- isoform) and human IGF1R (IGF-1R). (JPG 531 kb)

Supplementary Figure 3

Polypeptide fold for the IR monomer showing the relative arrangement of domains. (JPG 270 kb)

Supplementary Figure 4

Segment of difference electron density lying across the face of domain L1. (JPG 302 kb)

Supplementary Figure 5

Nucleotide and derived amino acid sequences of the light and heavy chain variable regions of the Fab from the anti-insulin receptor monoclonal antibody 83-7. (DOC 28 kb)

Supplementary Figure 6

Nucleotide and derived amino acid sequences of the light and heavy chain variable regions of the Fab from the anti-insulin receptor monoclonal antibody 83-14. (DOC 38 kb)

Supplementary Figure 7

McKern et al. Supplementary Figure S7. Cartoon of insulin binding to membrane-anchored IR. (JPG 4885 kb)

Supplementary Figure 8

Stereo images of 2Fo-Fc maps contoured at 1 σ contour level produced by BUSTER-TNT. (DOC 1833 kb)

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McKern, N., Lawrence, M., Streltsov, V. et al. Structure of the insulin receptor ectodomain reveals a folded-over conformation. Nature 443, 218–221 (2006). https://doi.org/10.1038/nature05106

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