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Symmetric and asymmetric receptor conformation continuum induced by a new insulin

Abstract

Cone snail venoms contain a wide variety of bioactive peptides, including insulin-like molecules with distinct structural features, binding modes and biochemical properties. Here, we report an active humanized cone snail venom insulin with an elongated A chain and a truncated B chain, and use cryo-electron microscopy (cryo-EM) and protein engineering to elucidate its interactions with the human insulin receptor (IR) ectodomain. We reveal how an extended A chain can compensate for deletion of B-chain residues, which are essential for activity of human insulin but also compromise therapeutic utility by delaying dissolution from the site of subcutaneous injection. This finding suggests approaches to developing improved therapeutic insulins. Curiously, the receptor displays a continuum of conformations from the symmetric state to a highly asymmetric low-abundance structure that displays coordination of a single humanized venom insulin using elements from both of the previously characterized site 1 and site 2 interactions.

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Fig. 1: Alignment of insulin sequences.
Fig. 2: Activities of Vh-Ins analogs based on cone snail venoms containing extended A-chain sequences.
Fig. 3: The symmetric Vh-Ins-HSLQ–receptor structure.
Fig. 4: Receptor binding affinity.
Fig. 5: Conformational heterogeneity in Vh-Ins-HSLQ–receptor reconstructions.
Fig. 6: Activity of Vh-Ins-HALQ relative to human insulin.

Data availability

RNA-sequencing data have been deposited into the GenBank Nucleotide Database (accession numbers MW091321, MW091322, MW091323 and MW091324). Coordinates of the refined atomic models have been deposited in the Protein Data Bank (‘head’: 7MQO; ‘whole’: 7MQR; ‘asymmetric’: 7MQS). The associated cryo-EM maps have been deposited in the Electron Microscopy Data Bank (‘head’: EMD-23949; ‘whole’: EMD-23950; ‘asymmetric’: EMD-23951). Raw cryo-EM movies are available on the Electron Microscopy Public Image Archive (accession code: EMPIAR-10736). Other data are available from the corresponding authors upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank B.M. Olivera for cone snail collection and identification and insightful discussions, P. Shen for advice with structure determination and editing, D. Timm for EM screening and data collection at the University of Utah Electron Microscopy Core Laboratory and Paula Flórez Salcedo for the illustration of the Conus kinoshitai shell. The support and resources from the Center for High Performance Computing and the High Throughput Genomics Core Facility at the University of Utah are gratefully acknowledged. Financial support was provided by the National Institutes of Health NIDDK (DK120430 to D.H.-C.C., DK127268 to C.P.H. and DK118082 to S.J.F.), NIGMS (GM125001 to D.H.-C.C.), Juvenile Diabetes Research Foundation (5-CDA-2018-572-A-N to D.H.-C.C. and 1-INO-2017-441-A-N to H.S.-H.), German Federal Ministry of Education and Research (BMBF) grant to the German Center for Diabetes Research (DZD e.V. to Ü.C.), Deutsche Forschungsgemeinschaft (DFG 251981924–TRR 83 to Ü.C. and DFG 347368302 to Ü.C. and T.G.) and the Australian National Health and Medical Research Council (APP1143546 to M.C.L. and B.E.F.). Support of M.C.L.’s research is also made possible at WEHI through Victorian State Government Operational Infrastructure Support and the Australian NHMRC Independent Research Institutes Infrastructure Support Scheme. H.S.-H. acknowledges fellowship support from the Villum Foundation (19063) and the Carlsberg Foundation (CF19-0445).

Author information

Authors and Affiliations

Authors

Contributions

X.X. designed, synthesized and purified insulin analogs. A.B. prepared cryo-EM samples, processed cryo-EM data and modeled atomic coordinates with input from I.B.S. A.B. performed analytical ultracentrifugation with D.E. J.H.K., Y.W.Z. and X.X. performed pAkt-based activity assays. J.G.M. and M.C.L. performed the ITC experiments. H.L.S. screened cryo-EM samples and collected data sets. T.G., G.O.A. and Ü.C. expressed and purified the IR ectodomain and performed nanoDSF experiments and western blots to assess signal transduction in Hep-G2 cells. C.D., A.M. and B.E.F. performed signal transduction western blots and DNA synthesis assays in L6 myoblasts. R.A. and S.J.F. performed in vivo glucose response assays. X.X., A.B., H.S.-H., C.P.H. and D.H.-C.C. interpreted data, generated figures and wrote the manuscript with significant input from M.C.L., Ü.C., I.B.S. and T.G. All authors reviewed and edited the manuscript.

Corresponding authors

Correspondence to Helena Safavi-Hemami, Christopher P. Hill or Danny Hung-Chieh Chou.

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A patent application related to this work was filed by the University of Utah.

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Extended data

Extended Data Fig. 1 Precursor sequence alignment of venom insulins identified in this study.

Canonical arrangement of preproinsulins with N-terminal signal sequences (purple) followed by the B chain (blue), C-peptide region (black) and A chain (green). The signal sequence, C peptide(s), and additional black-colored residues are predicted to be cleaved during post-translational processing.

Extended Data Fig. 2 AKT phosphorylation activity of Vh-Ins-HTLQ and related analogs.

NIH 3T3 cells overexpressing IR-B were stimulated with insulin analogs and pAkt was quantified using a homogeneous time-resolved fluorescence assay. Error bars (s.e.m. of 4 biological replicates) are shown when larger than the symbols. Two substitutions on the B chain, GluB10 and LeuB20, were found to increase the relative activity of Vh-Ins-HTLQ. These substitutions were subsequently included in later stages of design of Vh-Ins molecules.

Source data

Extended Data Fig. 3 Vh-Ins-HSLQ at site 2.

Density is shown around Vh-Ins-HSLQ. Vh-Ins-HSLQ green, with Vh-Ins mutated residues relative to native human insulin shown in orange. Receptor FnIII-1 domain, purple. The only Vh-specific residue that approaches receptor at site 2 is GluB10, which has poor density. Nearby receptor side chains lack density but are shown explicitly for illustrative purposes.

Extended Data Fig. 4 Activity of Vh-Ins analogs with single-residue substitutions in the extended A-chain residues (A21-A24).

NIH 3T3 cells overexpressing IR-B were stimulated with insulin analogs and pAkt was quantified using a homogeneous time-resolved fluorescence assay. Error bars (s.e.m. of 4 biological replicates) are shown when larger than the symbols.

Source data

Extended Data Fig. 5 Vh-Ins-HALQ binding to IR and IGF-1R ectodomains.

a, NanoDSF monitoring of intrinsic protein fluorescence to determine the thermal conformational stability of IR-ECD (top) or IGF1R-ECD (bottom) in the presence of respective ligands in four-times molar excess. Apo-IR-ECD displays two detectable unfolding transitions Tmlow and Tmhigh at 59.2 °C and 63.2 °C, respectively (Supplementary Table 5). The presence of Vh-Ins-HALQ leads to a decrease in Tmlow to 56.3 °C indicating conformational changes induced by ligand binding similarly to insulin (Tmlow = 54.3 °C). Apo-IGF1R-ECD displays a single transition temperature Tmhigh, while binding to hIGF-I leads to an additional melting transition at 57.4 °C. No significant changes in unfolding transitions were observed for IR-ECD in the presence of hIGF-I or for IGF1R-ECD in the presence of Vh-Ins-HALQ or hIns as compared to the respective ligand-free ectodomains. b, MST with IR-ECD (left) and IGF1R-ECD (right) to determine dissociation constants of binding to respective ligands (Supplementary Table 6; n = 3, error bars show standard deviations).

Source data

Extended Data Fig. 6 Comparison of the two receptor protomers in the asymmetric conformation against previously reported structures.

Left, Vh-Ins:IR asymmetric state apolike protomer (blue) vs apo IR (PDBs 4ZXB). Right, the second Vh-Ins:IR protomer (pink) vs insulin-bound receptor (6PXV) following alignment on L1, CR, L2, and FnIII-1 domains.

Extended Data Fig. 7 Vh-Ins-HALQ signal transduction in Hep-G2 cells.

Signal transduction in Hep-G2 hepatoblastoma cells induced by Vh-Ins-HALQ and hIns at 10 or 50 nM was assessed by Western blot and densitometry (4-6 biological replicates for each condition). Phosphorylation-specific antibodies were used to detect phosphorylated IR (Y1150/115), IGF1R (Y980), AKT1 (S473), and MEK-1/2 (S217/221). Relative intensities of specific protein bands were normalized to the GAPDH loading control and then to the respective signal after 5 min of insulin treatment.

Source data

Extended Data Fig. 8 SV-AUC c(s) analysis of insulin analogs.

a, DOI (des octapeptide insulin) and hIns (human insulin) controls at a concentration of 100 µg/ml and 775 µg/ml respectively in phosphate buffer (137 mM NaCl, 2.7 mM KCl, 5.3 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4). b, Vh-Ins-HSLQ and Lispro both at 500 µg/ml in sterile insulin diluent (16 mg/ml glycerol, 1.6 mg/ml m-cresol, 0.65 mg/ml phenol, 3.8 mg/ml Na2HPO4, pH 7.4). c-d, Data fit and residuals for DOI, hIns, Vh-Ins-HSLQ and lispro, respectively. For clarity, some scans are omitted from the figures shown but all scans were used for the c(s) analysis. The interval between the scans shown in each panel is ~9 minutes.

Source data

Extended Data Fig. 9 SV-AUC of DOI and hIns in sterile diluent.

a, c(s) analysis of DOI and hIns in sterile diluent. For reference the Vh-Ins-HSLQ trace from Fig. S11 is shown. b-c, data fit and residuals for DOI and hIns in sterile diluent, respectively. hIns shows increasing concentrations at higher radii in early scans, indicative of aggregation during the experiment.

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–6, Note and Tables 1–6.

Reporting Summary

Supplementary Video 1

A series of volumes derived from 3D variability analysis show a range of flexible motion in one of the two receptor protomers. Models were built into the conformational extremes and used to create a series of molecular motions that were fit into the experimental density. In the asymmetric extreme, three Vh-Ins molecules are apparent in the complex, including one in a composite site 1/site 2 position. As the receptor moves toward the symmetric state, L1 and CR move away from their position near the composite site, and the separate density for both site 1- and site 2-bound Vh-Ins becomes apparent.

Source data

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Unprocessed western blots.

Source Data Extended Data Fig. 2

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Source Data Extended Data Fig. 9

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Xiong, X., Blakely, A., Kim, J.H. et al. Symmetric and asymmetric receptor conformation continuum induced by a new insulin. Nat Chem Biol 18, 511–519 (2022). https://doi.org/10.1038/s41589-022-00981-0

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