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Insulin signaling controls metabolic homeostasis. Here, we report the cryo-EM structure of full-length insulin receptor (IR) and insulin complex in the active state. This structure unexpectedly reveals that maximally four insulins can bind the ‘T’-shaped IR dimer at four distinct sites related by 2-fold symmetry. Insulins 1 and 1’ bind to sites 1 and 1’, formed by L1 of one IR protomer and α-CT and FnIII-1 of the other. Insulins 2 and 2’ bind to sites 2 and 2’ on FnIII-1 of each protomer. Mutagenesis and cellular assays show that both sites 1 and 2 are required for optimal insulin binding and IR activation. We further identify a homotypic FnIII-2–FnIII-2 interaction in mediating the dimerization of membrane proximal domains in the active IR dimer. Our results indicate that binding of multiple insulins at two distinct types of sites disrupts the autoinhibited apo-IR dimer and stabilizes the active dimer.

Purpose Comparison of the dissociation kinetics of rapid-acting insulins lispro, aspart, glulisine and human insulin under physiologically relevant conditions. Methods Dissociation kinetics after dilution were monitored directly in terms of the average molecular mass using combined static and dynamic light scattering. Changes in tertiary structure were detected by near-UV circular dichroism. Results Glulisine forms compact hexamers in formulation even in the absence of Zn 2+ . Upon severe dilution, these rapidly dissociate into monomers in less than 10 s. In contrast, in formulations of lispro and aspart, the presence of Zn 2+ and phenolic compounds is essential for formation of compact R6 hexamers. These slowly dissociate in times ranging from seconds to one hour depending on the concentration of phenolic additives. The disadvantage of the long dissociation times of lispro and aspart can be diminished by a rapid depletion of the concentration of phenolic additives independent of the insulin dilution. This is especially important in conditions similar to those after subcutaneous injection, where only minor dilution of the insulins occurs. Conclusion Knowledge of the diverging dissociation mechanisms of lispro and aspart compared to glulisine will be helpful for optimizing formulation conditions of rapid-acting insulins.

Insulin/IGF signaling (IIS) is a highly conserved pathway essential for physiological regulation from yeast to mammals. In Drosophila melanogaster , a single insulin-like receptor (dmIR) interacts with various insulin-like peptides (DILPs), leading to diverse signaling and functional outcomes. However, the mechanisms by which different DILPs result in varied receptor activation and biological responses remain unclear. Here, we determine the cryo-electron microscopy (cryo-EM) structures of dmIR in complex with three DILPs: DILP1, DILP2, and DILP5. Our structural analyses reveal that each DILP induces distinct conformations of dmIR: the dmIR/DILP5 complex adopts the Ƭ -shaped asymmetric conformation with three bound DILP5 molecules; the dmIR/DILP2 complex displays the Γ -shaped asymmetric conformation with a single bound DILP2 molecule; and the dmIR/DILP1 complex shows both a Γ -shaped asymmetric conformation and a symmetric conformation that resembles a T-shape with a splayed stem. Functional assays demonstrate that the efficacy of DILP-mediated dmIR activation differs, with DILP5 inducing higher levels of receptor autophosphorylation, followed by DILP2 and DILP1. Together, these findings suggest that the distinct interactions between dmIR and DILPs dictate specific patterns of receptor activation. Drosophila has a single insulin/IGF receptor (dmIR) that responds to various s insulin-like peptides (DILP ligands). Here, authors determined the cryo-EM structures of dmIR bound with three different DILP ligands and revealed how distinct ligands fine-tune dmIR signaling.

Insulin receptor (IR) signaling controls multiple facets of animal physiology. Maximally four insulins bind to IR at two distinct sites, termed site-1 and site-2. However, the precise functional roles of each binding event during IR activation remain unresolved. Here, we showed that IR incompletely saturated with insulin predominantly forms an asymmetric conformation and exhibits partial activation. IR with one insulin bound adopts a Γ-shaped conformation. IR with two insulins bound assumes a Ƭ -shaped conformation. One insulin binds at site-1 and another simultaneously contacts both site-1 and site-2 in the Ƭ -shaped IR dimer. We further show that concurrent binding of four insulins to sites-1 and -2 prevents the formation of asymmetric IR and promotes the T-shaped symmetric, fully active state. Collectively, our results demonstrate how the synergistic binding of multiple insulins promotes optimal IR activation. Through structural and functional analyses, this work defines the molecular mechanisms underlying the activation of the insulin receptor (IR) involving multisite insulin binding, paving the way for the eventual therapeutic intervention for diseases caused by aberrant activation of IR.

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.Insights from structural biology lead to the development of mini-Ins—a human des-octapeptide insulin analog that is monomeric and has receptor binding affinity and in vitro and in vivo activities comparable to those of human insulin.

The three-dimensional structure of the insulin–insulin receptor complex has proved elusive, confounded by the complexity of producing the receptor protein; here is the first glimpse of the interaction between insulin and its primary binding site on the insulin receptor, a view based on four crystal structures of insulin bound to truncated insulin receptor complexes. A first look at insulin–receptor binding Despite more than three decades of research, the three-dimensional structure of the complex between insulin and its receptor has proved elusive, confounded by the complexity of producing the receptor protein. Here is a first glimpse of the interaction between insulin and its primary binding site on the insulin receptor — a view based on four crystal structures of insulin bound to truncated receptor complexes. Surprisingly, the bulk of the interaction with the receptor leucine-rich repeat ligand-binding domain is indirect and mediated through a helical peptide segment (αCT) that is provided by the alternate insulin partner in the receptor dimer. Insulin receptor signalling has a central role in mammalian biology, regulating cellular metabolism, growth, division, differentiation and survival 1 , 2 . Insulin resistance contributes to the pathogenesis of type 2 diabetes mellitus and the onset of Alzheimer’s disease 3 ; aberrant signalling occurs in diverse cancers, exacerbated by cross-talk with the homologous type 1 insulin-like growth factor receptor (IGF1R) 4 . Despite more than three decades of investigation, the three-dimensional structure of the insulin–insulin receptor complex has proved elusive, confounded by the complexity of producing the receptor protein. Here we present the first view, to our knowledge, of the interaction of insulin with its primary binding site on the insulin receptor, on the basis of four crystal structures of insulin bound to truncated insulin receptor constructs. The direct interaction of insulin with the first leucine-rich-repeat domain (L1) of insulin receptor is seen to be sparse, the hormone instead engaging the insulin receptor carboxy-terminal α-chain (αCT) segment, which is itself remodelled on the face of L1 upon insulin binding. Contact between insulin and L1 is restricted to insulin B-chain residues. The αCT segment displaces the B-chain C-terminal β-strand away from the hormone core, revealing the mechanism of a long-proposed conformational switch in insulin upon receptor engagement. This mode of hormone–receptor recognition is novel within the broader family of receptor tyrosine kinases 5 . We support these findings by photo-crosslinking data that place the suggested interactions into the context of the holoreceptor and by isothermal titration calorimetry data that dissect the hormone–insulin receptor interface. Together, our findings provide an explanation for a wealth of biochemical data from the insulin receptor and IGF1R systems relevant to the design of therapeutic insulin analogues.

Background Due to population aging, an increasing number of elderly patients with diabetes use insulin. It is therefore important to investigate the characteristics of new insulins in this population. Faster-acting insulin aspart (faster aspart) is insulin aspart (IAsp) in a new formulation with faster absorption. This study investigated the pharmacological properties of faster aspart in elderly subjects with type 1 diabetes mellitus (T1DM). Methods In a randomised, double-blind, two-period crossover trial, 30 elderly (≥65 years) and 37 younger adults (18–35 years) with T1DM received single subcutaneous faster aspart or IAsp dosing (0.2 U/kg) and underwent an euglycaemic clamp (target 5.5 mmol/L) for up to 12 h. Results The pharmacokinetic and pharmacodynamic time profiles were left-shifted for faster aspart versus IAsp. In each age group, onset of appearance occurred approximately twice as fast (~3 min earlier) and early exposure (area under the concentration–time curve [AUC] for serum IAsp from time zero to 30 min [AUC IAsp,0-30 min ]) was greater (by 86% in elderly and 67% in younger adults) for faster aspart than for IAsp. Likewise, onset of action occurred 10 min faster in the elderly and 9 min faster in younger adults, and early glucose-lowering effect (AUC for the glucose infusion rate [GIR] from time zero to 30 min [AUC GIR,0-30 min ]) was greater (by 109%) for faster aspart than for IAsp in both age groups. Total exposure (AUC IAsp,0-t ) and the maximum concentration ( C max ) for faster aspart were greater (by 30 and 28%, respectively) in elderly than in younger adults. No age group differences were seen for the total (AUC GIR,0-t ) or maximum (GIR max ) glucose-lowering effect. Conclusion This study demonstrated that the ultra-fast pharmacological properties of faster aspart are similar in elderly subjects and younger adults with T1DM. ClinicalTrials.gov Identifier: NCT02003677.

The development of effective and safe insulin analogs remains pivotal in advancing diabetes management. This study addresses the limitations of existing insulin therapies by introducing insulin lisargine, a novel long-acting insulin analog that resolves impurity formation associated with trypsin cleavage in glargine insulin. Insulin lisargine is characterized by glycine substitution at A21 and the addition of lysine and arginine at B31 and B32, respectively. High-performance liquid chromatography (HPLC) and mass spectrometry confirmed its high purity and precise molecular weight. X-ray crystallography at 2.0 Å resolution revealed structural features closely resembling human insulin, crucial for optimizing drug formulations and understanding receptor interactions.In vivo experiments demonstrated that insulin lisargine exhibits superior glucose-lowering effects compared to glargine insulin (Lantus). At a dosage of 1.5 IU/kg, lisargine achieved glucose-lowering effects equivalent to glargine in normal rats. However, at 5 IU/kg, it significantly outperformed glargine in type 1 diabetic rats. Long-term safety assessments revealed a comparable safety profile between lisargine and glargine, with no significant toxicity observed. These findings position insulin lisargine as a promising candidate for diabetes management, offering enhanced blood glucose control, improved production efficiency, and reliable safety. The study’s findings provide a foundation for the development of more effective insulin analogs, addressing critical needs in diabetes therapy.

Structural elucidation and biochemical analysis of a cone snail insulin venom that binds and activates the human insulin receptor may permit design of ultrafast-acting insulin analogs for diabetes therapy. 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.

The production of recombinant insulin remains challenging, particularly in enhancing refolding efficiency and bioactivity. Mini-proinsulin analogs, which involve reducing the length of the C-peptide, offer potential improvements in insulin production. This study aims to evaluate mini-proinsulin analogs’ design and receptor binding dynamics to optimize recombinant insulin production in E. coli . Mini-proinsulin analogs were engineered by replacing the 33-residue C-peptide with a pentapeptide sequence to improve refolding. The three-dimensional structure of mini-proinsulin was predicted using AlphaFold and performed docking analysis of mini-proinsulin analogs to the insulin receptor using AutoDock Tools, with comparisons made to previously available NMR-determined analog and the native insulin-insulin receptor complex. Normal Mode Analyses (GNM and ANM) were performed in detail to assess binding dynamics. In silico analyses revealed that mini-proinsulin analogs closely replicate the structural features of native insulin and display receptor binding dynamics similar to native insulin, though they follow distinct receptor interaction paths. All analysis suggests that C-peptide mobility may contribute to the allosteric behavior observed in mini-proinsulin analogs during receptor interaction.