Abstract
Human insulin and its current therapeutic analogs all show propensity, albeit varyingly, to self-associate into dimers and hexamers, which delays their onset of action and makes blood glucose management difficult for people with diabetes. Recently, we described a monomeric, insulin-like peptide in cone-snail venom with moderate human insulin-like bioactivity. Here, with insights from structural biology studies, we report the development of mini-Ins—a human des-octapeptide insulin analog—as a structurally minimal, full-potency insulin. Mini-Ins is monomeric and, despite the lack of the canonical B-chain C-terminal octapeptide, has similar receptor binding affinity to human insulin. Four mutations compensate for the lack of contacts normally made by the octapeptide. Mini-Ins also has similar in vitro insulin signaling and in vivo bioactivities to human insulin. The full bioactivity of mini-Ins demonstrates the dispensability of the PheB24–PheB25–TyrB26 aromatic triplet and opens a new direction for therapeutic insulin development.
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Data availability
Coordinates and structures factors for the structures presented here have been deposited in the Protein Data Bank as follows: Cons-Ins–G1-bound Fv83-7-bound μIR, PDB 6VEQ; human insulin-bound Fv83-7-bound μIR, PDB 6VEP and mini-Ins, PDB 6VET. Supplementary Methods and Source Data for Figs. 4a,b,d, 5b–d, 6 and 7a,b and Extended Data Figs. 2 and 3 are available with the paper online.
Change history
12 June 2020
A Correction to this paper has been published: https://doi.org/10.1038/s41594-020-0460-2
References
Safavi-Hemami, H. et al. Specialized insulin is used for chemical warfare by fish-hunting cone snails. Proc. Natl Acad. Sci. USA 112, 1743–1748 (2015).
Menting, J. G. et al. A minimized human insulin-receptor-binding motif revealed in a Conus geographus venom insulin. Nat. Struct. Mol. Biol. 23, 916–920 (2016).
Carpenter, F. H. Relationship of structure to biological activity of insulin as revealed by degradative studies. Am. J. Med. 40, 750–758 (1966).
Gradel, A. K. J. et al. Factors affecting the absorption of subcutaneously administered insulin: effect on variability. J. Diabetes Res. 2018, 1205121 (2018).
Maikawa, C. L. et al. Stable monomeric insulin formulations enabled by supramolecular pegylation of insulin analogues. Adv. Ther. 3, 1900094 (2020).
Kang, S., Brange, J., Burch, A., Vølund, A. & Owens, D. R. Subcutaneous insulin absorption explained by insulin’s physicochemical properties. Evidence from absorption studies of soluble human insulin and insulin analogues in humans. Diabetes Care 14, 942–948 (1991).
Kristensen, C., Andersen, A. S., Østergaard, S., Hansen, P. H. & Brandt, J. Functional reconstitution of insulin receptor binding site from non-binding receptor fragments. J. Biol. Chem. 277, 18340–18345 (2002).
Uchikawa, E., Choi, E., Shang, G., Yu, H. & Bai, X.-C. Activation mechanism of the insulin receptor revealed by cryo-EM structure of the fully liganded receptor-ligand complex. Elife 8, e48630 (2019).
Scapin, G. et al. Structure of the insulin receptor–insulin complex by single-particle cryo-EM analysis. Nature 556, 122–125 (2018).
Weis, F. et al. The signalling conformation of the insulin receptor ectodomain. Nat. Commun. 9, 4420 (2018).
Gutmann, T. et al. Cryo-EM structure of the complete and ligand-saturated insulin receptor ectodomain. J. Cell Biol. 219, e201907210 (2020).
Menting, J. G. et al. Protective hinge in insulin opens to enable its receptor engagement. Proc. Natl Acad. Sci. USA 111, E3395–E3404 (2014).
Menting, J. G. et al. How insulin engages its primary binding site on the insulin receptor. Nature 493, 241–245 (2013).
De Meyts, P. & Whittaker, J. Structural biology of insulin and IGF1 receptors: implications for drug design. Nat. Rev. Drug Discov. 1, 769–783 (2002).
Kiselyov, V. V., Versteyhe, S., Gauguin, L. & De Meyts, P. Harmonic oscillator model of the insulin and IGF1 receptors’ allosteric binding and activation. Mol. Sys. Biol. 5, 243 (2009).
Lawrence, C. F. et al. Insulin mimetic peptide disrupts the primary binding site of the insulin receptor. J. Biol. Chem. 291, 15473–15481 (2016).
Nakagawa, S. H. & Tager, H. S. Role of the phenylalanine B25 side chain in directing insulin interaction with its receptor. Steric and conformational effects. J. Biol. Chem. 261, 7332–7341 (1986).
Pandyarajan, V. et al. Aromatic anchor at an invariant hormone-receptor interface: function of insulin residue B24 with application to protein design. J. Biol. Chem. 289, 34709–34727 (2014).
Kikuchi, K. et al. Studies on the biological activity of degraded insulins and insulin fragments. J. Biol. Chem. 255, 9281–9288 (1980).
Schwartz, G. P., Burke, G. T. & Katsoyannis, P. G. A superactive insulin: [B10-aspartic acid]insulin(human). Proc. Natl Acad. Sci. USA 84, 6408–6411 (1987).
Kaarsholm, N. C. et al. Engineering stability of the insulin monomer fold with application to structure-activity relationships. Biochemistry 32, 10773–10778 (1993).
Kristensen, C. et al. Alanine scanning mutagenesis of insulin. J. Biol. Chem. 272, 12978–12983 (1997).
Thayer, W. P., Kraft, J. R., Tompkins, S. M., Moore, J. C. T. & Jensen, P. E. Assessment of the role of determinant selection in genetic control of the immune response to insulin in H-2b mice. J. Immunol. 163, 2549–2554 (1999).
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).
Schäffer, L. A model for insulin binding to the insulin receptor. Eur. J. Biochem. 221, 1127–1132 (1994).
Smith, G. D., Pangborn, W. A. & Blessing, R. H. The structure of T6 human insulin at 1.0 Å resolution. Acta Crystallogr. D. 59, 474–482 (2003).
Ahorukomeye, P. et al. Fish-hunting cone snail venoms are a rich source of minimized ligands of the vertebrate insulin receptor. Elife 8, e41574 (2019).
De Meyts, P. Insulin/receptor binding: the last piece of the puzzle? What recent progress on the structure of the insulin/receptor complex tells us (or not) about negative cooperativity and activation. Bioessays 37, 389–397 (2015).
Glendorf, T. et al. Systematic evaluation of the metabolic to mitogenic potency ratio for B10-substituted insulin analogues. PLoS ONE 7, e29198 (2012).
Cara, J. F., Mirmira, R. G., Nakagawa, S. H. & Tager, H. S. An insulin-like growth factor I/insulin hybrid exhibiting high potency for interaction with the type I insulin-like growth factor and insulin receptors of placental plasma membranes. J. Biol. Chem. 265, 17820–17825 (1990).
Kurtzhals, P. et al. Correlations of receptor binding and metabolic and mitogenic potencies of insulin analogs designed for clinical use. Diabetes 49, 999–1005 (2000).
Laskowski, R. A. SURFNET: a program for visualizing molecular surfaces, cavities, and intermolecular interactions. J. Mol. Graph. 13, 323–330 (1995).
Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).
Aragão, D. et al. MX2: a high-flux undulator microfocus beamline serving both the chemical and macromolecular crystallography communities at the Australian Synchrotron. J. Synchrotron Radiat. 25, 885–891 (2018).
Kabsch, W. Integration, scaling, space-group assignment and post-refinement. Acta Crystallogr. D Biol. Crystallogr. 66, 133–144 (2010).
McCoy, A. J. et al. PHASER crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).
Winn, M. D., Isupov, M. N. & Murshudov, G. N. Use of TLS parameters to model anisotropic displacements in macromolecular refinement. Acta Crystallogr. D Biol. Crystallogr. 57, 122–133 (2001).
Bricogne, G. et al. BUSTER v.2.10 (Global Phasing Ltd, 2011).
McKern, N. M. et al. Structure of the insulin receptor ectodomain reveals a folded-over conformation. Nature 443, 218–221 (2006).
Berry, M. B. et al. Structure of an anti-HIV monoclonal Fab antibody fragment specific to a gp120 C-4 region peptide. Proteins 45, 281–282 (2001).
Teplyakov, A. et al. On the domain pairing in chimeric antibodies. Mol. Immunol. 47, 2422–2426 (2010).
Celikel, R., Ruggeri, Z. M. & Varughese, K. I. von Willebrand factor conformation and adhesive function is modulated by an internalized water molecule. Nat. Struct. Biol. 7, 881–884 (2000).
Evans, P. R. & Murshudov, G. N. How good are my data and what is the resolution? Acta Crystallogr. D Biol. Crystallogr. 69, 1204–1214 (2013).
Webb, B. & Sali, A. Comparative protein structure modeling using MODELLER. Curr. Protoc. Bioinformatics 47, 5.6.1–5.6.32 (2014).
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).
Abraham, M. J. et al. GROMACS: high performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX 1–2, 19–25 (2015).
Guvench, O. et al. CHARMM additive all-atom force field for carbohydrate derivatives and its utility in polysaccharide and carbohydrate-protein modeling. J. Chem. Theory Comput. 7, 3162–3180 (2011).
Best, R. B. et al. Optimization of the additive CHARMM all-atom protein force field targeting improved sampling of the backbone ϕ, ψ and side-chain χ1 and χ2 dihedral angles. J. Chem. Theory Comput. 8, 3257–3273 (2012).
Hess, B. P-LINCS: a parallel linear constraint solver for molecular simulation. J. Chem. Theory Comput. 4, 116–122 (2008).
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).
Rajapaksha, H. & Forbes, B. E. Ligand-binding affinity at the insulin receptor isoform-A and subsequent IR-A tyrosine phosphorylation kinetics are important determinants of mitogenic biological outcomes. Front. Endocrinol. 6, 107 (2015).
Acknowledgements
X.X. is a Juvenile Diabetes Research Foundation Postdoctoral Fellow. N.A.S. acknowledges receipt of an Australian Research Training Scholarship. R.S.N acknowledges fellowship support from the Australian National Health and Medical Research Council. Part of this work was undertaken using resources from the National Computational Infrastructure, which is supported by the Australian Government and provided through Intersect Australia Ltd, and through the HPC-GPGPU Facility, which was established with the assistance of a Linkage Infrastructure, Equipment and Facilities grant (LE170100200). We thank the CSIRO Protein Production Facility for the production under contract of cIR485, the precursor of IR310.T. Crystallization screening was undertaken at the CSIRO Collaborative Crystallisation Centre (www.csiro.au/C3), Melbourne, Australia. This research was undertaken in part using the MX2 beamline at the Australian Synchrotron, part of the Australian Nuclear Science and Technology Organisation, and made use of the ACRF detector. We thank M. Margetts for production of the heavy and light chain fragments of Fv83-7. This work is supported by NIDDK (DK120430 to D.H.C.), NIGMS (GM125001 to D.H.C.), the Juvenile Diabetes Research Foundation (5-CDA-2018-572-A-N to D.H.C. and 1-INO-2017-441-A-N to H.S.H.), the Australian National Health and Medical Research Council (NHMRC) Project grant nos. APP1143546 (to M.C.L., R.S.N., B.J.S., B.E.F. and D.H.C.) and APP1099595 (to M.C.L.). M.C.L.’s research is also made possible at The Walter and Eliza Hall Institute of Medical Research through Victorian State Government Operational Infrastructure Support and the Australian NHMRC Independent Research Institutes Infrastructure Support Scheme.
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Contributions
X.X., J.G.M., R.S.N., H.S.-H., B.O., M.C.L. and D.H.-C.C. designed the study. M.C.L. and D.H.-C.C. wrote the manuscript with input from all authors. M.M.D. and J.G. generated Con-Ins–G1 analogs and M.M.D. performed related in vitro assays with assistance from G.G., and N.A.S. and B.J.S. performed modeling studies. C.D. and B.E.F. performed receptor binding and signaling studies. R.A. and S.J.F. performed in vivo bioactivity assays. X.W. and X.H. performed antibody response assays. C.A.M. and R.S.N. performed analytical ultracentrifugation studies. J.G.M and M.C.L performed crystallographic studies, and J.G.M. produced protein.
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M.C.L.’s laboratory has a funded Agreement with Eli Lilly and Company (USA) to conduct research not connected to this publication. Patents associated with part of this work were licensed to Monolog LLC, which aims to develop new fast-acting insulin analogs.
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Extended data
Extended Data Fig. 1 Stereo views of sample (2mFobs-DFcalc) electron density for the structures presented in the manuscript.
(a) L1 domain residues 32-35 within monomer 1 of the Con-Ins-G1-bound μIR + Fv 83-7 crystal structure. (b) L1 domain residues 32-35 within monomer 1 of the human-insulin- bound μIR + Fv 83-7 crystal structure. (c) B-chain residues 14-16 within monomer 1 of the mini-Ins crystal structure. All maps are contoured at the 1.0 σ level.
Extended Data Fig. 2 Sedimentation equilibrium analysis of mini-Ins.
Sedimentation equilibrium analysis of mini-Ins was performed at 35,000 rpm with the best fit (curves) to a single species of apparent mass 5080 ± 45 Da. The molecular weight of mini-Ins is 5067. Detailed procedure can be found in ref. 2. Source data are available with the paper online.
Extended Data Fig. 3 Antibody response of 21-day immunization of bovine insulin, human insulin and mini-Ins.
Data are the average of 4 independent animals. Error bar represents S.E.M. Source data are available with the paper online.
Extended Data Fig. 4 Isothermal titration calorimetry.
Representative ITC thermograms for the titration against IR485 + IR-A704–719 αCT peptide of (a) mini-Ins; (b) hIns; (c) Con-Ins G1 and (d) human DOI.
Extended Data Fig. 5 Separation of insulin and IR amino acid pairs at the secondary binding site during 1 ns MD simulation.
(a) Distance between GluB10 carboxylate carbon (Cδ) and Arg539 guanyl carbon (Cζ). This salt pair remain closely associated (~4 Å) throughout the simulation. (b) Distance between HisA8 imidazole Nε2 nitrogen and Asp574 carboxylate carbon. (c) Distance between ArgA9 guanyl carbon and Glu575 carboxylate carbon (Cγ): this salt bridge forms (separation ~4 Å) following ~6ns MD. The salt bridge is observed to dissociate and reform several times throughout the simulation. Dissociation of this interaction correlates with increase in separation of the HisA8-Asp574 pair, reflecting mobility in the Phe572-to-Tyr579 loop of the FnIII-1.
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Xiong, X., Menting, J.G., Disotuar, M.M. et al. A structurally minimized yet fully active insulin based on cone-snail venom insulin principles. Nat Struct Mol Biol 27, 615–624 (2020). https://doi.org/10.1038/s41594-020-0430-8
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DOI: https://doi.org/10.1038/s41594-020-0430-8
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