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Synergistic activation of the insulin receptor via two distinct sites

Abstract

Insulin receptor (IR) signaling controls multiple facets of animal physiology. Maximally four insulins bind to IR at two distinct sites, termed site-1 and site-2. However, the precise functional roles of each binding event during IR activation remain unresolved. Here, we showed that IR incompletely saturated with insulin predominantly forms an asymmetric conformation and exhibits partial activation. IR with one insulin bound adopts a Γ-shaped conformation. IR with two insulins bound assumes a Ƭ-shaped conformation. One insulin binds at site-1 and another simultaneously contacts both site-1 and site-2 in the Ƭ-shaped IR dimer. We further show that concurrent binding of four insulins to sites-1 and -2 prevents the formation of asymmetric IR and promotes the T-shaped symmetric, fully active state. Collectively, our results demonstrate how the synergistic binding of multiple insulins promotes optimal IR activation.

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Fig. 1: Binding to IR by insulin site-1 and site-2 mutants.
Fig. 2: Structure of IR with insulin bound only to site-2.
Fig. 3: Structures of IR with insulin LeuA13R bound only to site-1.
Fig. 4: Structures of IR with insulin LeuB17R bound only to site-1 and functional importance of site-2 insulin binding.
Fig. 5: Mixture of insulin site-1 and site-2 mutants promotes the formation of T-shaped IR dimer and activates IR signaling.
Fig. 6: Structures of IR/insulin WT complex at subsaturated insulin concentrations.
Fig. 7: Working models for insulin-induced IR activation.

Data availability

All reagents generated in this study are available with a completed Materials Transfer Agreement. All cryo-EM maps and models reported in this work have been deposited into EMDB/PDB database, under the entry ID: EMD-25188/PDB 7SL1, EMD-25189/PDB 7SL2, EMD-25190/PDB 7SL3, EMD-25191/PDB 7SL4, EMD-25192/PDB 7SL6, EMD-25193/PDB 7SL7, EMD-25428/PDB 7STH, EMD-25249/PDB 7STI, EMD-25430/7STJ and EMD-25431/7STK. Source data are provided with this paper.

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Acknowledgements

Cryo-EM data were collected at the University of Texas Southwestern Medical Center (UTSW) Cryo-Electron Microscopy Facility, funded in part by the Cancer Prevention and Research Institute of Texas (CPRIT) Core Facility Support Award RP170644. We thank D. Stoddard for facility access. This work is supported in part by grants from the National Institutes Health (R01GM136976 to X.-C.B., R35GM142937, P30DK063608 and UL1TR001873 to E.C., AG061829 to M.H.B.S and R35GM130289 to X.Z), the Welch foundation (I-1944 to X.-C.B. and I-1702 to X.Z.), CPRIT (RP160082 to X.-C.B.), the MCDB Neurodegenerative Disease Fund (to M.H.B.S.), the T. Curtius Peptide Facility (to M.H.B.S.) and the Alice Bohmfalk Charitable (to E.C.). X.-C.B. and X.Z. are Virginia Murchison Linthicum Scholars in Medical Research at UTSW. This paper is dedicated to the memory of Dr. Misha Plam.

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Authors

Contributions

M.H.B.S., E.C. and X.-C.B. designed and supervised the research. All the authors performed the research. J.P.M., X.Z., M.H.B.S., E.C. and X.-C.B. analyzed the data. J.P.M., M.H.B.S., E.C. and X.-C.B. wrote the paper with input from the other authors.

Corresponding authors

Correspondence to Michael H. B. Stowell, Eunhee Choi or Xiao-chen Bai.

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Nature Structural & Molecular Biology thanks Stevan Hubbard, John Rubinstein and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Florian Ullrich, in collaboration with the Nature Structural & Molecular Biology team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Domains of insulin receptor (IR) and activities of insulin analogs in primary mouse hepatocytes.

a. Domains and disulfide connectivity of IR. L1 and L2, leucine rich domains 1 and 2; CR, cysteine rich domain; F1, F2 and F3, fibronectin III (FnIII) domains; ID, insert in FnIII-2 domain; TM, transmembrane domain; TK, tyrosine kinase domain. b. HPLC traces for each of the insulins synthesized and utilized for both functional and structural studies. c. MS1 spectra of the purified insulins in B analyzed in the Orbitrap mass analyzer. d. Binding of insulin WT and site1 mutant (IleA2A;ValA3A) labeled with Alexa Fluor 488 to purified IR WT in the indicated conditions (Mean ± SD, WT, n = 9 independent experiments; IleA2A;ValA3A, n = 3). Significance calculated using two-tailed student t-test; **p < 0.01 and ***p < 0.001 (The exact p values are provided in the source data). e. Insulin competition-binding assay for isolated FnIII-1 domain and insulin WT and mutants (ValA3E and LeuB17R) (Mean ± SD, n = 3). f. Insulin-induced IR autophosphorylation in 293FT cells expressing IR wild-type (WT). Cells were treated with the indicated insulin WT or site-2 mutants for 10 min. The IR autophosphorylation levels were assessed by quantitative western blotting with a phospho-tyrosine (pY) IRβ antibody. Expression levels of IRβ were monitored by anti-Myc blotting against the C-terminal Myc-tag. g. Quantification of the western blot data shown in e (Mean ± SD). Each experiment was repeated four times. Significance calculated using two-tailed student t-test; *p < 0.05; **p < 0.01, ***p < 0.001, and ****p < 0.0001 (The exact p values are provided in the source data). Uncropped images for all blots and gels are available as source data.

Source data

Extended Data Fig. 2 Purification of the full-length mouse insulin receptor (IR).

a. A representative size-exclusion chromatography of IR. b. The peak fractions were combined and visualized on SDS-PAGE by Coomassie staining, in the absence or presence of dithiothreitol (DTT). Most of IR was processed into α-chain (IRα) and β-chain (IRβ). This experiment was repeated for 10 times independently with similar results.

Extended Data Fig. 3 Cryo-EM analysis of the IR-insulin site-1 mutant (ValA3E) complex.

a. Representative electron micrograph and 2D class averages of the IR-insulin site-1 mutant (ValA3E) complex. Scale bar: 200 Å. This experiment was repeated for 2,879 times independently with similar results. b. Unsharpened cryo-EM map colored by local resolution. c. The gold-standard Fourier Shell Correlation (FSC) curve for the cryo-EM map shown in Fig. 2a. d. Flowchart of cryo-EM data processing.

Extended Data Fig. 4 Cryo-EM analysis of the IR-insulin site-2 mutant (LeuA13R) complex.

a. Representative electron micrograph and 2D class averages of the IR-insulin site-2 mutant (LeuA13R) complex. Scale bar: 200 Å. This experiment was repeated for 3,783 times independently with similar results. b. Unsharpened cryo-EM map colored by local resolution. c. The gold-standard Fourier Shell Correlation (FSC) curve for the cryo-EM map shown in Fig. 3a. d. Flowchart of cryo-EM data processing.

Extended Data Fig. 5 Close-up view of asymmetric IR/insulin LeuA13R complex.

a. Overall view of asymmetric IR/insulin LeuA13R complex in two orthogonal views. b. The close-up view of the contact of site-1 bound insulin LeuA13R at FnIII-1 domain in the asymmetric IR/insulin LeuA13R complex. The location of this interaction in the asymmetric dimer is indicated by a blue box in a. c. The close-up view of the binding of a dimeric insulin at asymmetric IR/insulin LeuA13R complex. The location of this interaction in the asymmetric dimer is indicated by a red box in a.

Extended Data Fig. 6 Cryo-EM analysis of the IR-insulin site-2 mutant (LeuB17R) complex.

a. Representative electron micrograph and 2D class averages of the IR-insulin site-2 mutant (LeuB17R) complex. Scale bar: 200 Å. This experiment was repeated for 3,995 times independently with similar results. b. Unsharpened cryo-EM map colored by local resolution. c. The gold-standard Fourier Shell Correlation (FSC) curve for the cryo-EM map shown in Fig. 4a. d. Flowchart of cryo-EM data processing.

Extended Data Fig. 7 Insulin binding to site-2 of IR facilitates IR activation and signaling.

a. IR signaling in 293FT cells expressing IR wild-type (WT). Cells were treated with the 10 nM insulin WT and site-2 mutants for the indicated times. Cell lysates were blotted with the indicated antibodies. b. Quantification of the western blot data shown in a (Mean ± SEM, For pY/IR, n = 7 independent experiments; pAKT/AKT, n = 4, pERK/ERK, n = 6). Significance calculated using two-tailed student t-test; *p < 0.05; **p < 0.01, ***p < 0.001, and ****p < 0.0001 (The exact p values are provided in the source data). c. IR signaling in primary mouse hepatocytes treated with the indicated concentrations of insulin for 10 min. Cell lysates were blotted with the indicated antibodies. Quantification of the western blot data shown in Fig. 4e. d. IR signaling in primary mouse hepatocytes treated with 10 nM insulin for the indicated times. Cell lysates were blotted with the indicated antibodies. Quantification of the western blot data shown in Fig. 4g. Uncropped images for all blots and gels are available as source data.

Source data

Extended Data Fig. 8 Cryo-EM analysis of the IR-insulin site-1 (ValA3E) and site-2 (LeuA13R) mutants complex.

a. Representative electron micrograph and 2D class averages of the IR-insulin site-1 (ValA3E) and site-2 (LeuA13R) mutants complex. Scale bar: 200 Å. This experiment was repeated for 4,895 times independently with similar results. b. Unsharpened cryo-EM map colored by local resolution. c. The gold-standard Fourier Shell Correlation (FSC) curve for the cryo-EM map shown in Fig. 5a, b. d. Flowchart of cryo-EM data processing.

Extended Data Fig. 9 Cryo-EM analysis of the IR-insulin WT complex at subsaturated insulin concentrations.

a. Representative electron micrograph and 2D class averages of the IR-insulin WT complex at subsaturated insulin concentrations. Scale bar: 200 Å. This experiment was repeated for 3,635 times independently with similar results. b. Unsharpened cryo-EM maps colored by local resolution. c. The gold-standard Fourier Shell Correlation (FSC) curve for the cryo-EM map shown in Fig. 6a-d. d. Flowchart of cryo-EM data processing.

Extended Data Fig. 10 Close-up view of insulin, α-CT in asymmetric IR dimer and membrane proximal domains in asymmetric and symmetric IR dimer.

a. The close-up view of the contact of site-2 bound insulin at α-CT in the asymmetric IR/insulin complex. b. Close-up view of α-CT in 1 and 2 insulins bound asymmetric IR dimer. c. Superposition of the hybrid sites between the Ƭ-shaped asymmetric conformations 1 and 2, showing the rotation of insulin around the α-CT and two different insulin binding modes. d. Close-up view of the membrane proximal domains in asymmetric and symmetric IR dimer. e. Superposition between the membrane proximal domains in asymmetric and symmetric IR dimer.

Supplementary information

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Source Data Extended Data Fig. 1

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Statistical source data.

Source Data Extended Data Fig. 7

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Li, J., Park, J., Mayer, J.P. et al. Synergistic activation of the insulin receptor via two distinct sites. Nat Struct Mol Biol 29, 357–368 (2022). https://doi.org/10.1038/s41594-022-00750-6

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