Cryo-EM structure of the complete and ligand-saturated insulin receptor ectodomain

Gutmann, Theresia; Schäfer, Ingmar B.; Poojari, Chetan; Brankatschk, Beate; Vattulainen, Ilpo; Strauss, Mike; Coskun, Ünal

doi:10.1101/679233

Cited by 13 publications

(29 citation statements)

References 67 publications

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“…This indicates that studied insulin residues might not be involved in direct interactions with site 2 of receptors. Recently, two studies (42, 43) were published showing cryo-EM maps of the IR extracellular domains with insulin bound to a newly identified binding site that was proposed as a new site 2 and should interact with the insulin residues studied in this work. These new findings support the hypothesis of initial docking and transient interactions between insulin and IR.…”

Section: Discussionmentioning

confidence: 92%

“…It is not excluded that future advances in X-ray crystallography or cryo-EM methodology will decipher structures of such hypothetical transient protein hormone complexes and reveal complex mechanisms of receptor activation by the hormones. In this context, during the preparation and revision of this manuscript, two studies (42, 43) were published showing cryo-EM maps of the IR extracellular domains with four insulins bound. Two of these insulins are positioned as shown previously by Scapin et al (21) but the binding site for the other two insulins is located in the FnIII-1 (or FnIII-1′) domain and was not detected previously.…”

Section: Discussionmentioning

confidence: 99%

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Mutations at hypothetical binding site 2 in insulin and insulin-like growth factors 1 and 2 result in receptor- and hormone-specific responses

Macháčková

Mlčochová

Potalitsyn

et al. 2019

Journal of Biological Chemistry

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Information on how insulin and insulin-like growth factors 1 and 2 (IGF-1 and -2) activate insulin receptors (IR-A and -B) and the IGF-1 receptor (IGF-1R) is crucial for understanding the difference in the biological activities of these peptide hormones. Cryo-EM studies have revealed that insulin uses its binding sites 1 and 2 to interact with IR-A and have identified several critical residues in binding site 2. However, mutagenesis studies suggest that Ile-A10, Ser-A12, Leu-A13, and Glu-A17 also belong to insulin's site 2. Here, to resolve this discrepancy, we mutated these insulin residues and the equivalent residues in IGFs. Our findings revealed that equivalent mutations in the hormones can result in differential biological effects and that these effects can be receptor-specific. We noted that the insulin positions A10 and A17 are important for its binding to IR-A and IR-B and IGF-1R and that A13 is important only for IR-A and IR-B binding. The IGF-1/IGF-2 positions 51/50 and 54/53 did not appear to play critical roles in receptor binding, but mutations at IGF-1 position 58 and IGF-2 position 57 affected the binding. We propose that IGF-1 Glu-58 interacts with IGF-1R Arg-704 and belongs to IGF-1 site 1, a finding supported by the NMR structure of the less active Asp-58–IGF-1 variant. Computational analyses indicated that the aforementioned mutations can affect internal insulin dynamics and inhibit adoption of a receptor-bound conformation, important for binding to receptor site 1. We provide a molecular model and alternative hypotheses for how the mutated insulin residues affect activity.

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Section: Discussionmentioning

confidence: 92%

Section: Discussionmentioning

confidence: 99%

Mutations at hypothetical binding site 2 in insulin and insulin-like growth factors 1 and 2 result in receptor- and hormone-specific responses

Macháčková

Mlčochová

Potalitsyn

et al. 2019

Journal of Biological Chemistry

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“…During the revision of this manuscript, another study deposited in the preprint server bioRxiv also showed a low-resolution cryo-EM map of the IR extracellular domain with four insulins bound at sites 1 and 2 (Gutmann et al, 2019). The 3:1 or 4:1 stoichiometry of insulin binding to the IR dimer and the two types of binding sites are consistent with previous IR–insulin binding assays that indicated the coexistence of high- and low-affinity insulin-binding sites (Schäffer, 1994).…”

Section: Resultsmentioning

confidence: 99%

Activation mechanism of the insulin receptor revealed by cryo-EM structure of the fully liganded receptor–ligand complex

Uchikawa

Choi

Shang

et al. 2019

eLife

143

276

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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.

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“…S1 ). A significant number of the residues that have been previously shown to be involved in insulin:IR or IGF-1:IGF1R interaction [ [28] , [29] , [30] , [31] , [32] , [33] ] are either conserved or conservatively substituted in dcVILPs ( Figure 1 A, Table 1 ). Structural studies have suggested that the mature IR or IGF1R can bind two insulin or IGF-1 molecules with high affinity, effectively crosslinking two binding subsites on IR/IGF1R named as Site 1 and Site 2 [ [28] , [29] , [30] , [34] , [35] , [36] ].…”

Section: Resultsmentioning

confidence: 99%

Characterization of viral insulins reveals white adipose tissue-specific effects in mice

Marchand

Noh

Páníková

et al. 2021

Molecular Metabolism

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Objective Members of the insulin/insulin-like growth factor (IGF) superfamily are well conserved across the evolutionary tree. We recently showed that four viruses in the Iridoviridae family possess genes that encode proteins highly homologous to human insulin/IGF-1. Using chemically synthesized single-chain (sc), i.e., IGF-1-like, forms of the viral insulin/IGF-1-like peptides (VILPs), we previously showed that they can stimulate human receptors. Because these peptides possess potential cleavage sites to form double chain (dc), i.e., more insulin-like, VILPs, in this study, we have characterized dc forms of VILPs for Grouper iridovirus (GIV), Singapore grouper iridovirus (SGIV) and Lymphocystis disease virus-1 (LCDV-1) for the first time. Methods The dcVILPs were chemically synthesized. Using murine fibroblast cell lines overexpressing insulin receptor (IR-A or IR-B) or IGF1R, we first determined the binding affinity of dcVILPs to the receptors and characterized post-receptor signaling. Further, we used C57BL/6J mice to study the effect of dcVILPs on lowering blood glucose. We designed a 3-h dcVILP in vivo infusion experiment to determine the glucose uptake in different tissues. Results GIV and SGIV dcVILPs bind to both isoforms of human insulin receptor (IR-A and IR-B) and to the IGF1R, and for the latter, show higher affinity than human insulin. These dcVILPs stimulate IR and IGF1R phosphorylation and post-receptor signaling in vitro and in vivo. Both GIV and SGIV dcVILPs stimulate glucose uptake in mice. In vivo infusion experiments revealed that while insulin (0.015 nmol/kg/min) and GIV dcVILP (0.75 nmol/kg/min) stimulated a comparable glucose uptake in heart and skeletal muscle and brown adipose tissue, GIV dcVILP stimulated 2-fold higher glucose uptake in white adipose tissue (WAT) compared to insulin. This was associated with increased Akt phosphorylation and glucose transporter type 4 (GLUT4) gene expression compared to insulin in WAT. Conclusions Our results show that GIV and SGIV dcVILPs are active members of the insulin superfamily with unique characteristics. Elucidating the mechanism of tissue specificity for GIV dcVILP will help us to better understand insulin action, design new analogs that specifically target the tissues and provide new insights into their potential role in disease.

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Cryo-EM structure of the complete and ligand-saturated insulin receptor ectodomain

Cited by 13 publications

References 67 publications

Mutations at hypothetical binding site 2 in insulin and insulin-like growth factors 1 and 2 result in receptor- and hormone-specific responses

Mutations at hypothetical binding site 2 in insulin and insulin-like growth factors 1 and 2 result in receptor- and hormone-specific responses

Activation mechanism of the insulin receptor revealed by cryo-EM structure of the fully liganded receptor–ligand complex

Characterization of viral insulins reveals white adipose tissue-specific effects in mice

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