916 VOLUME 23 NUMBER 10 OCTOBER 2016 NATURE STRUCTURAL & MOLECULAR BIOLOGY ARTICLES Animal venoms are a rich source of valuable pharmacological tools, drug leads and therapeutics. Venom components are typically small proteins that are highly stable in the extracellular environment, read- ily bioavailable and extremely specific to their physiological target; these properties have been shaped through millions of years of evolu- tion to offer a streamlined role in predation, defense and deterrence. Several venom-derived therapeutics have been approved by the US Food and Drug Administration, and many more are in preclinical studies and clinical trials 1,2 . The recent discovery 3 of specialized venom insulins in Conus geographus that are divergent from endogenous molluscan insulins but strikingly similar to fish (and therefore human) insulin provides a unique opportunity to investigate the pharmacological potential of these fast-acting natural proteins that have evolved to specifically affect glucose homeostasis. In healthy humans, insulin (hIns) is stored in pancreatic beta cells as a hexamer consisting of three insulin dimers held together by two central zinc ions; the insulin monomer itself con- sists of a 21-residue A chain and a 30-residue B chain, which are cross- linked by two disulfide bridges (Cys A7 -Cys B7 and Cys A20 -Cys B19 ) and a third disulfide bridge within the A chain (Cys A6 -Cys A11 ) 4 (Fig. 1a). Insulin hexamer-to-monomer conversion is crucial to its bioavailabil- ity and can lead to a delay in glucose control after injection into dia- betic patients. Insulin administration typically involves a combination of a rapid-acting preprandial insulin and a longer-acting basal insu- lin 5 . Rapid-acting insulins contain amino acid substitutions that are deleterious to insulin multimerization 5 but retain the aromatic triplet Phe B24 -Phe B25 -Tyr B26 , despite its role in dimerization, because Phe B24 is critical for activity. Phe B24 lies immediately C terminal to a type 1 β-turn formed by residues Gly B20 -Glu B21 -Arg B22 -Gly B23 , and both the triplet and the type 1 β-turn are highly conserved in vertebrate insulins. Attempts to shorten the C terminus of insulin B chain to abolish self-association have resulted in near-complete loss of activity. For example, des-octapeptide(B23–B30) insulin (DOI), a monomeric analog, retains less than 0.1% of the wild-type bioactivity 6 . By contrast, the C. geographus insulin Con-Ins G1 lacks any equivalent of Arg B22 through Thr B30 of hIns (Fig. 1a) but retains the canonical disulfide-bonding pattern of vertebrate insulins. We thus hypothesized that (i) Cons-Ins G1 is likely to be active against the human insulin receptor (hIR), given the high level of sequence conservation between fish and human insulin receptor, and that (ii) Con-Ins G1 contains structural elements that act as a surrogate for the critical hIns residue Phe B24 . We present here a biochemical, bio- physical and crystallographic investigation of Con-Ins G1 that tested these hypotheses. Our findings provide a basis for the design of a new generation of ultrarapid-acting insulins. RESULTS Synthesis of native and selenocysteine forms of Cons-Ins G1 We synthesized 1.5 mg of Con-Ins G1 through solid-phase peptide synthesis methods based on Fmoc chemistry, determined the purity of the final product to be 89% and 80% by reverse-phase (RP) HPLC and capillary electrophoresis, respectively, and confirmed the mass 1 Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia. 2 Department of Biology, University of Utah, Salt Lake City, Utah, USA. 3 Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia. 4 Department of Biochemistry, University of Utah, Salt Lake City, Utah, USA. 5 La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia. 6 Sentia Medical Sciences, San Diego, California, USA. 7 Department of Medical Biochemistry, Flinders University, Bedford Park, South Australia, Australia. 8 Department of Biology, University of Copenhagen, Copenhagen, Denmark. 9 Department of Medical Biology, University of Melbourne, Parkville, Victoria, Australia. 10 These authors jointly supervised this work. Correspondence should be addressed to M.C.L. (lawrence@wehi.edu.au). Received 25 May; accepted 16 August; published online 12 September 2016; doi:10.1038/nsmb.3292 A minimized human insulin-receptor-binding motif revealed in a Conus geographus venom insulin John G Menting 1 , Joanna Gajewiak 2 , Christopher A MacRaild 3 , Danny Hung-Chieh Chou 4 , Maria M Disotuar 4 , Nicholas A Smith 5 , Charleen Miller 6 , Judit Erchegyi 6 , Jean E Rivier 6 , Baldomero M Olivera 2 , Briony E Forbes 7 , Brian J Smith 5 , Raymond S Norton 3 , Helena Safavi-Hemami 2,8,10 & Michael C Lawrence 1,9,10 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 Phe B24 is mitigated. These findings may facilitate efforts to design ultrarapid-acting therapeutic insulins. npg © 2016 Nature America, Inc. All rights reserved. npg © 2016 Nature America, Inc. All rights reserved.