Abstract
We report the crystal structure of two variants of Drosophila melanogaster insulin-like peptide 5 (DILP5) at a resolution of 1.85 Å. DILP5 shares the basic fold of the insulin peptide family (T conformation) but with a disordered B-chain C terminus. DILP5 dimerizes in the crystal and in solution. The dimer interface is not similar to that observed in vertebrates, i.e. through an anti-parallel β-sheet involving the B-chain C termini but, in contrast, is formed through an anti-parallel β-sheet involving the B-chain N termini. DILP5 binds to and activates the human insulin receptor and lowers blood glucose in rats. It also lowers trehalose levels in Drosophila. Reciprocally, human insulin binds to the Drosophila insulin receptor and induces negative cooperativity as in the human receptor. DILP5 also binds to insect insulin-binding proteins. These results show high evolutionary conservation of the insulin receptor binding properties despite divergent insulin dimerization mechanisms.
Highlights
We report the crystal structure of two variants of Drosophila melanogaster insulin-like peptide 5 (DILP5) at a resolution of 1.85 A
The same basic fold is shared for all molecules in the superfamily whose structure is known; the B domain contains a single ␣-helix that lies across the two ␣-helices of the A domain [15] and two canonical disulfide bridges that connect the A-and Bchains, whereas an intrachain disulfide bridge is present in the A-chain
We report here the first crystal structure of invertebrate insulins expressed from cloned cDNAs, namely two variants of DILP5, DB and C4, that differ by the absence or presence of an Asp-Phe-Arg sequence extension at the N terminus of the A-chain
Summary
Production of Recombinant Proteins—The cDNA encoding dilp was obtained by RT-PCR from D. melanogaster (OreR) ovaries mRNA. Crystallization and Structure Determination—Details of proteins crystallization, data collection, and processing and refinement statistics are given in the supplemental information. Crystallization of the proteins was carried out in hanging-drop vapor-diffusion experiments by mixing 1 l of protein (9 mg/ml) and 1 l of reservoir solution (20% PEG 4000) Both variants C4 and DB crystallized in the same space group P43212 with cell parameters a ϭ b ϭ 40.45 Å, and c ϭ 45.24 Å for DILP5-C4 and a ϭ b ϭ 39.79 Å, and c ϭ 45.32 Å for DILP5-DB. IM9 or S2 cells were incubated with [125I] human insulin (20,000 cpm/ml) and increasing concentrations of unlabeled ligand for 1 h for S2 cells and 2.5 h for IM9 cells at 15 °C, pH 7.6. To obtain a dose-response curve for negative cooperativity, duplicate aliquots were diluted 40-fold in the presence of increasing concentrations of unlabeled ligand for 30 min at 15 °C [28]. Plates were covered and incubated overnight at 37 °C, and optical density was measured at 340 nm
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
