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A minimized human insulin-receptor-binding motif revealed in a Conus geographus venom insulin


Insulins in the venom of certain fish-hunting cone snails facilitate prey capture by rapidly inducing hypoglycemic shock. One such insulin, Conus geographus G1 (Con-Ins G1), is the smallest known insulin found in nature and lacks the C-terminal segment of the B chain that, in human insulin, mediates engagement of the insulin receptor and assembly of the hormone's hexameric storage form. Removal of this segment (residues B23–B30) in human insulin results in substantial loss of receptor affinity. Here, we found that Con-Ins G1 is monomeric, strongly binds the human insulin receptor and activates receptor signaling. Con-Ins G1 thus is a naturally occurring B-chain-minimized mimetic of human insulin. Our crystal structure of Con-Ins G1 reveals a tertiary structure highly similar to that of human insulin and indicates how Con-Ins G1's lack of an equivalent to the key receptor-engaging residue PheB24 is mitigated. These findings may facilitate efforts to design ultrarapid-acting therapeutic insulins.

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Figure 1: Characterization of Con-Ins G1.
Figure 2: Three-dimensional structure of Con-Ins G1.
Figure 3: Molecular model of Con-Ins G1 in the context of the primary insulin-binding site of the human insulin receptor.
Figure 4: Detail of Con-Ins G1 residues proposed to interact with the primary binding site of hIR.

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R.S.N. acknowledges fellowship support from the National Health and Medical Research Council of Australia (NHMRC). N.A.S. acknowledges receipt of an Australian Postgraduate Award scholarship. This work was supported in part by National Institutes of Health grants GM 48677 (to B.M.O. and J.E.R., a subcontractor at Sentia Medical Sciences), by NHMRC Project Grant APP1058233 (to M.C.L.) and by the Utah Science and Technology Initiative (USTAR, to D.H.-C.C.). H.S.-H. acknowledges fellowship support from the European Commission (CONBIOS 330486). Aspects of this work were made possible through Victorian State Government Operational Infrastructure Support, the Australian Government NHMRC IRIISS and a pilot grant from the University of Utah Diabetes and Metabolism Center. We thank A. Morrione (Thomas Jefferson University) for providing cells. Part of this research was undertaken on the MX2 beamline at the Australian Synchrotron (Victoria, Australia).

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Authors and Affiliations



J.G.M. and M.C.L. performed crystallography; M.C.L., R.S.N., H.S.-H. and B.J.S. directed research; B.M.O., H.S.-H., R.S.N. and M.C.L. designed the study; B.E.F. and D.H.-C.C. performed experiments and analyzed data; J.G., C.M. and J.E. synthesized peptides and analyzed data; M.M.D. performed experiments; C.A.M. performed ultracentrifugation experiments, N.A.S. and B.J.S. performed computer modeling, J.E.R. supervised peptide synthesis, and M.C.L., R.S.N., H.S.-H. and B.J.S. wrote the manuscript.

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Correspondence to Michael C Lawrence.

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M.C.L.'s and D.H.C.'s research activities are partially funded by Sanofi (Germany).

Integrated supplementary information

Supplementary Figure 1 Arrangement of Con-Ins G1 monomers around the crystallographic four-fold axis (stereo).

The four Con-Ins G1 monomers coordinate an apparent single off-axis sulfate molecules (centre), modelled with unrestrained coordinates into a relatively featureless blob (not shown) of difference electron density lying on the crystallographic four-fold axis. The ion forms part of a charge-compensated cluster comprising the amino-terminal group of GlyA1 and a side-chain carboxylate of GlaA4 from each Con-Ins G1 monomer. The Con-Ins G1 A chains are in pink and the B chains in light blue.

Supplementary Figure 2 Amino acid sequence and RP-HPLC profile of fully oxidized Con-Ins G1.

The left-hand panel shows the amino acid sequence of fully-oxidized Con-Ins G1 (top: A chain; bottom, B chain). O: hydroxyproline, γ: γ-carboxyglutamate, *: amidated C-terminus. The right-hand panel shows the RP-HPLC profile. RP-HPLC conditions are: C18 Vydac RP-HPLC column, linear gradient ranging from 15 to 45% of solvent B in 30 min with 1 mL / min flow rate monitored at 220 nm.

Supplementary Figure 3 Amino acid sequence and RP-HPLC profile of fully oxidized PTM-free sCon-Ins G1.

The left-hand panel shows the amino acid sequence of fully-oxidized PTM-free sCon-Ins G1 (top: A chain; bottom, B chain). Residues in bold indicate site of mutation; U: selenocysteine, *: amidated C-terminus. The right-hand panel shows the RP-HPLC profile. RP-HPLC conditions are: C18 Vydac RP-HPLC column, linear gradient ranging from 15 to 45% of solvent B in 30 min with 1 mL / min flow rate monitored at 220 nm.

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Menting, J., Gajewiak, J., MacRaild, C. et al. A minimized human insulin-receptor-binding motif revealed in a Conus geographus venom insulin. Nat Struct Mol Biol 23, 916–920 (2016).

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