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

AbstractGlucose 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.

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

The Journal of Cell Biology ◽

10.1083/jcb.201907210 ◽

2019 ◽

Vol 219 (1) ◽

Cited By ~ 14

Author(s):

Theresia Gutmann ◽

Ingmar B. Schäfer ◽

Chetan Poojari ◽

Beate Brankatschk ◽

Ilpo Vattulainen ◽

...

Keyword(s):

Insulin Receptor ◽

Binding Sites ◽

Receptor Site ◽

Insulin Binding ◽

Dynamics Simulation ◽

Structural Basis ◽

Combined Approach ◽

Comprehensive Description ◽

Structure Based Drug Design ◽

Receptor Interactions

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.

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Structural basis by which a recessive mutation in the alpha-subunit of the insulin receptor affects insulin binding.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)36552-3 ◽

1994 ◽

Vol 269 (21) ◽

pp. 14912-14918

Author(s):

M. Taouis ◽

R. Levy-Toledano ◽

P. Roach ◽

S.I. Taylor ◽

P. Gorden

Keyword(s):

Insulin Receptor ◽

Insulin Binding ◽

Alpha Subunit ◽

Structural Basis ◽

Recessive Mutation

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Molecular Modeling and Bioinformatics Analysis of Drug-Receptor Interactions in the System Formed by Glargine, Its Metabolite M1, the Insulin Receptor, and the IGF1 Receptor

Bioinformatics and Biology Insights ◽

10.1177/11779322211046403 ◽

2021 ◽

Vol 15 ◽

pp. 117793222110464

Author(s):

Margarita González-Beltrán ◽

Claudio Gómez-Alegría

Keyword(s):

Growth Factor ◽

Insulin Receptor ◽

Structural Basis ◽

Insulin Like Growth Factor ◽

Receptor Models ◽

Bioinformatics Tools ◽

Receptor Interactions ◽

Factor Type ◽

Drug Receptor

Introduction: Insulin and insulin-like growth factor type 1 (IGF1) regulate multiple physiological functions by acting on the insulin receptor (IR) and insulin-like growth factor type 1 receptor (IGF1R). The insulin analog glargine differs from insulin in three residues (GlyA21, ArgB31, ArgB32), and it is converted to metabolite M1 (lacks residues ArgB31 and ArgB32) by in vivo processing. It is known that activation of these receptors modulates pathways related to metabolism, cell division, and growth. Though, the structures and structural basis of the glargine interaction with these receptors are not known. Aim: To generate predictive structural models, and to analyze the drug/receptor interactions in the system formed by glargine, its metabolite M1, IR, and IGF1R by using bioinformatics tools. Methods: Ligand/receptor models were built by homology modeling using SWISSMODEL, and surface interactions were analyzed using Discovery Studio® Visualizer. Target and hetero target sequences and appropriate template structures were used for modeling. Results: Our glargine/IR and metabolite M1/IR models showed an overall symmetric T-shaped conformation and full occupancy with four ligand molecules. The glargine/IR model revealed that the glargine residues ArgB31 and ArgB32 fit in a hydrophilic region formed by the α-chain C-terminal helix (αCT) and the cysteine-rich region (CR) domain of this receptor, close to the CR residues Arg270-Arg271-Gln272 and αCT residue Arg717. Regarding IGF1R, homologous ligand/receptor models were further built assuming that the receptor is in a symmetrical T-shaped conformation and is fully occupied with four ligand molecules, similar to what we described for IR. Our glargine/IGF1R model showed the interaction of the glargine residues ArgB31 and ArgB32 with Glu264 and Glu305 in the CR domain of IGF1R. Conclusion: Using bioinformatics tools and predictive modeling, our study provides a better understanding of the glargine/receptor interactions.

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Insulin Receptor: 3D Reconstruction from Darkfield Stem Images, Structural Interpretation and Functional Model

Microscopy and Microanalysis ◽

10.1017/s1431927600015361 ◽

1999 ◽

Vol 5 (S2) ◽

pp. 408-409

Author(s):

F.P. Ottensmeyer ◽

R.Z.-T. Luo ◽

A.B. Fernandes ◽

D. Benia ◽

C.C. Yip

Keyword(s):

Growth Factor ◽

Insulin Receptor ◽

Tyrosine Kinases ◽

Quaternary Structure ◽

Functional Model ◽

Growth Factor Receptor ◽

Insulin Binding ◽

Dark Field ◽

Bovine Insulin ◽

Structural Basis

We have reconstructed the three-dimensional quaternary structure of the complete 480 kDa insulin receptor (IR), complexed with NanoGold-labelled insulin, via sets of electron micrographs obtained by low-dose low-temperature dark field scanning transmission electron microscopy (STEM).Insulin binding to IR in mammalian cell membranes is essential for its manifold effects such as glucose homeostasis, increased protein synthesis, growth, and development. IR belongs to the superfamily of transmembrane receptor tyrosine kinases that include the monomeric epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR). In contrast, IR and its homologues IGF-1R (insulin-like growth factor 1 receptor) and IRR (insulin receptorrelated receptor) are sub-types of this family that are intrinsic disulfide-linked dimers of two αβ heterodimers. Monomeric receptor TKs are inactive, but are activated by ligand-induced dimerization that results in autophosphorylation. IR-like TKs are also inactive even though they are already dimeric, and are activated by ligand binding without further oligomerization. Insulin binding to the extracellular domain of IR results in autophosphorylation of specific tyrosines to initiate an intracellular signal transduction cascade. However, because the quaternary structure of IR is not known, the structural basis for the mechanism of IR activation by extracellular insulin binding has not been elucidated.The insulin receptor was purified from human placenta. Bovine insulin was derivatized with NanoGold at the B chain Phel, a location not directly involved in receptor binding. Binding of derivatized insulin to the purified receptor was reduced only slightly compared to binding of the native insulin.

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Insulin-like growth factor binding to the atypical insulin receptors of a human lymphoid-derived cell line (IM-9)

Biochemical Journal ◽

10.1042/bj2660737 ◽

1990 ◽

Vol 266 (3) ◽

pp. 737-742 ◽

Cited By ~ 16

Author(s):

H A Jonas ◽

A J Cox

Keyword(s):

Growth Factor ◽

Affinity Chromatography ◽

Insulin Receptor ◽

Binding Sites ◽

Insulin Binding ◽

Type I ◽

Insulin Like Growth Factor ◽

Igf Receptors ◽

Type I Igf Receptor ◽

Igf I

The cells of the IM-9 human lymphocyte-derived line contain a sub-population of insulin-binding sites whose immunological and hormone-binding characteristics closely resemble those of the atypical insulin-binding sites of human placenta. These binding sites, which have moderately high affinity for multiplication-stimulating activity [MSA, the rat homologue of insulin-like growth factor (IGF) II] and IGF-I, are identified on IM-9 cells by 125I-MSA binding. They account for approximately 30% of the total insulin-receptor population, and do not react with a monoclonal antibody to the type I IGF receptor (alpha IR-3). The relative concentrations of unlabelled insulin, MSA and IGF-I required to displace 50% of 125I-MSA from these binding sites (1:4.7:29 respectively) are maintained for cells, particulate membranes, Triton-solubilized membranes precipitated either by poly(ethylene glycol) or a polyclonal antibody (B-10) to the insulin receptor, and receptors purified by insulin affinity chromatography. Because the atypical insulin/MSA-binding sites outnumber the type I IGF receptors in IM-9 cells by approximately 10-fold, they also compete with the latter receptors for 125I-IGF-I binding. Thus 125I-IGF-I binding to IM-9 cells is inhibited by moderately low concentrations of insulin (relative potency ratios for insulin compared with IGF-I are approx. 1/14 to 1/4) and is partially displaced (65-80%) by alpha IR-3. When type I IGF receptors are blocked by alpha IR-3 or removed by B-10 immunoprecipitation or insulin affinity chromatography, the hormone-displacement patterns for 125I-IGF-I binding resemble those of the atypical insulin/MSA-binding sites.

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Polylysine increases the number of insulin binding sites in soluble insulin receptor preparations.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)47382-1 ◽

1991 ◽

Vol 266 (26) ◽

pp. 17369-17375

Author(s):

Y. Biener ◽

Y. Zick

Keyword(s):

Insulin Receptor ◽

Binding Sites ◽

Insulin Binding ◽

Soluble Insulin ◽

Soluble Insulin Receptor

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Isolation of functional alpha beta heterodimers from the purified human placental alpha 2 beta 2 heterotetrameric insulin receptor complex. A structural basis for insulin binding heterogeneity.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)48180-x ◽

1987 ◽

Vol 262 (15) ◽

pp. 6939-6942 ◽

Cited By ~ 15

Author(s):

L J Sweet ◽

B D Morrison ◽

J E Pessin

Keyword(s):

Insulin Receptor ◽

Insulin Binding ◽

Structural Basis ◽

Receptor Complex ◽

Alpha Beta ◽

Beta 2

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Dominant Transmission of Insulin Resistance in a Type A Family Resulting From a Heterozygous Nonsense Mutation in the Insulin Receptor Gene and Associated With Decreased mRNA Level and Insulin Binding Sites

Diabetes ◽

10.2337/diab.46.11.1901 ◽

1997 ◽

Vol 46 (11) ◽

pp. 1901-1903 ◽

Cited By ~ 5

Author(s):

J. Magre ◽

C. Karayanni ◽

C. G. Hadjiathanasiou ◽

C. Desbois-Mouthon ◽

M. Meier ◽

...

Keyword(s):

Insulin Resistance ◽

Insulin Receptor ◽

Binding Sites ◽

Nonsense Mutation ◽

Receptor Gene ◽

Mrna Level ◽

Type A ◽

Insulin Binding ◽

Insulin Receptor Gene

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Identification of the ADPR binding pocket in the NUDT9 homology domain of TRPM2

The Journal of General Physiology ◽

10.1085/jgp.201611675 ◽

2017 ◽

Vol 149 (2) ◽

pp. 219-235 ◽

Cited By ~ 27

Author(s):

Peilin Yu ◽

Xiwen Xue ◽

Jianmin Zhang ◽

Xupang Hu ◽

Yan Wu ◽

...

Keyword(s):

Transient Receptor Potential ◽

Structural Model ◽

Receptor Potential ◽

Adenosine Diphosphate ◽

Binding Pocket ◽

Dynamics Simulation ◽

Structure Based Drug Design ◽

Transient Receptor Potential Melastatin ◽

Trpm2 Channel ◽

Transient Receptor

Activation of the transient receptor potential melastatin 2 (TRPM2) channel occurs during the response to oxidative stress under physiological conditions as well as in pathological processes such as ischemia and diabetes. Accumulating evidence indicates that adenosine diphosphate ribose (ADPR) is the most important endogenous ligand of TRPM2. However, although it is known that ADPR binds to the NUDT9 homology (NUDT9-H) domain in the intracellular C-terminal region, the molecular mechanism underlying ADPR binding and activation of TRPM2 remains unknown. In this study, we generate a structural model of the NUDT9-H domain and identify the binding pocket for ADPR using induced docking and molecular dynamics simulation. We find a subset of 11 residues—H1346, T1347, T1349, L1379, G1389, S1391, E1409, D1431, R1433, L1484, and H1488—that are most likely to directly interact with ADPR. Results from mutagenesis and electrophysiology approaches support the predicted binding mechanism, indicating that ADPR binds tightly to the NUDT9-H domain, and suggest that the most significant interactions are the van der Waals forces with S1391 and L1484, polar solvation interaction with E1409, and electronic interactions (including π–π interactions) with H1346, T1347, Y1349, D1431, and H1488. These findings not only clarify the roles of a range of newly identified residues involved in ADPR binding in the TRPM2 channel, but also reveal the binding pocket for ADPR in the NUDT9-H domain, which should facilitate structure-based drug design for the TRPM2 channel.

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