Glucose homeostasis and growth essentially depend on the hormone insulin engaging its receptor. Despite biochemical and structural advances, a fundamental contradiction has persisted in the current understanding of insulin ligand–receptor interactions. While biochemistry predicts two distinct insulin binding sites, 1 and 2, recent structural analyses have resolved only site 1. Using a combined approach of cryo-EM and atomistic molecular dynamics simulation, we present the structure of the entire dimeric insulin receptor ectodomain saturated with four insulin molecules. Complementing the previously described insulin–site 1 interaction, we present the first view of insulin bound to the discrete insulin receptor site 2. Insulin binding stabilizes the receptor ectodomain in a T-shaped conformation wherein the membrane-proximal domains converge and contact each other. These findings expand the current models of insulin binding to its receptor and of its regulation. In summary, we provide the structural basis for a comprehensive description of ligand–receptor interactions that ultimately will inform new approaches to structure-based drug design.

Using single-particle electron microscopy of the human insulin receptor reconstituted into nanosdiscs, Gutmann et al. show that ligand binding induces a conformational rearrangement in the receptor ectodomain that results in the dimerization of the transmembrane domains and receptor activation.

Lateral compositional and physicochemical heterogeneity is a ubiquitous feature of cellular membranes on various length scales, from molecular assemblies to micrometric domains. Segregated lipid domains of increased local order, referred to as rafts, are believed to be prominent features in eukaryotic plasma membranes; however, their exact nature (i.e. size, lifetime, composition, homogeneity) in live cells remains difficult to define. Here we present evidence that both synthetic and natural plasma membranes assume a wide range of lipid packing states with varying levels of molecular order. These states may be adapted and specifically tuned by cells during active cellular processes, as we show for stimulated insulin secretion. Most importantly, these states regulate both the partitioning of molecules between coexisting domains and the bioactivity of their constituent molecules, which we demonstrate for the ligand binding activity of the glycosphingolipid receptor GM1. These results confirm the complexity and flexibility of lipid-mediated membrane organization and reveal mechanisms by which this flexibility could be functionalized by cells.

How cold-blooded animals acclimate to temperature and what determines the limits of their viable temperature range are not understood. Here, we show that Drosophila alter their dietary preference from yeast to plants when temperatures drop below 15 C and that the different lipids present in plants improve survival at low temperatures. We show that Drosophila require dietary unsaturated fatty acids present in plants to adjust membrane fluidity and maintain motor coordination. Feeding on plants extends lifespan and survival for many months at temperatures consistent with overwintering in temperate climates. Thus, physiological alterations caused by a temperature-dependent dietary shift could help Drosophila survive seasonal temperature changes.

Lipidomes undergo permanent extensive remodeling, but how the turnover rate differs between lipid classes and molecular species is poorly understood. We employed metabolic N labeling and shotgun ultra-high-resolution mass spectrometry (sUHR) to quantify the absolute (molar) abundance and determine the turnover rate of glycerophospholipids and sphingolipids by direct analysis of total lipid extracts. sUHR performed on a commercial Orbitrap Elite instrument at the mass resolution of 1.35 × 10 (m/z 200) baseline resolved peaks of C isotopes of unlabeled and monoisotopic peaks ofN labeled lipids (Δm = 0.0063 Da). Therefore, the rate of metabolic N labeling of individual lipid species could be determined without compromising the scope, accuracy, and dynamic range of full-lipidome quantitative shotgun profiling. As a proof of concept, we employed sUHR to determine the lipidome composition and fluxes of 62 nitrogen-containing membrane lipids in human hepatoma HepG2 cells.

In the originally published version of the paper, the authors mistakenly substituted Figure S3 with an older version of Figure S4. The authors have now replaced it with the correct version of Figure S3, which is shown here and in the article online. The correct version of Figure S3 shows the diet and temperature dependence of sterol species, phospholipid classes, along with fatty acid double bonds and chain length. The original version of the paper was also missing three references: Kim et al., 2007;Liebisch et al., 2006;and Lö fgren et al., 2012. These are now provided here and in the article online. The authors apologize for any confusion these errors may have caused. . (2007). A two-photon fluorescent probe for lipid raft imaging: C-laurdan. ChemBioChem 8, 553-559. Liebisch, G., Binder, M., Schifferer, R., Langmann, T., Schulz, B., and Schmitz, G. (2006). High throughput quantification of cholesterol and cholesteryl ester by electrospray ionization tandem mass spectrometry (ESI-MS/MS). Biochim. Biophys. Acta 1761, 121-128. Lö fgren, L., Stå hlman, M., Forsberg, G.B., Saarinen, S., Nilsson, R., and Hansson, G.I. (2012). The BUME method: a novel automated chloroform-free 96-well total lipid extraction method for blood plasma.

Glucose homeostasis and growth essentially depend on the peptide hormone insulin engaging its receptor. Despite biochemical and structural advances, a fundamental contradiction has persisted in the current understanding of insulin ligand-receptor interactions. While biochemistry predicts two distinct insulin binding sites, 1 and 2, recent structural analyses have only resolved site 1.Using a combined approach of cryo-EM and atomistic molecular dynamics simulation, we determined the structure of the entire dimeric insulin receptor ectodomain saturated with four insulin molecules. Complementing the previously described insulin-site 1 interaction, we present the first view of insulin bound to the discrete insulin receptor site 2. Insulin binding stabilizes the receptor ectodomain in a T-shaped conformation wherein the membrane-proximal domains converge and contact each other. These findings expand the current models of insulin binding to its receptor and of its regulation. In summary, we provide the structural basis enabling a comprehensive description of ligand-receptor interactions that ultimately will inform new approaches to structure-based drug design. In briefA cryo-EM structure of the complete insulin receptor ectodomain saturated with four insulin ligands is reported. The structural model of the insulin-insulin receptor complex adopts a T-shaped conformation, reveals two additional insulin-binding sites potentially involved in the initial interaction of insulin with its receptor, and resolves the membrane proximal region.

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 a fully active humanized cone snail venom insulin with an elongated A chain and a truncated B chain, and use cryoelectron microscopy and protein engineering to elucidate its interactions with the human insulin receptor ectodomain. We reveal how an extended A chain can compensate for deletion of B-chain residues, which are essential for activity of native insulin but also compromise therapeutic utility by delaying the onset action, suggesting approaches to develop improved therapeutic insulins. Curiously, a receptor conformation present in low abundance adopts a highly asymmetric structure that displays novel coordination of a single humanized venom insulin using elements from both of the previously characterized site 1 and site 2 interactions.