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- PDB-7pg3: Low resolution Cryo-EM structure of the full-length insulin recep... -

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Basic information

Entry
Database: PDB / ID: 7pg3
TitleLow resolution Cryo-EM structure of the full-length insulin receptor bound to 3 insulin, conf 2
Components
  • (Insulin) x 2
  • Isoform Short of Insulin receptor
KeywordsMEMBRANE PROTEIN / Insulin / Receptor / Complex
Function / homology
Function and homology information


regulation of female gonad development / positive regulation of meiotic cell cycle / insulin-like growth factor II binding / positive regulation of developmental growth / male sex determination / insulin receptor complex / insulin-like growth factor I binding / exocrine pancreas development / positive regulation of protein-containing complex disassembly / dendritic spine maintenance ...regulation of female gonad development / positive regulation of meiotic cell cycle / insulin-like growth factor II binding / positive regulation of developmental growth / male sex determination / insulin receptor complex / insulin-like growth factor I binding / exocrine pancreas development / positive regulation of protein-containing complex disassembly / dendritic spine maintenance / cargo receptor activity / insulin binding / negative regulation of NAD(P)H oxidase activity / neuronal cell body membrane / adrenal gland development / PTB domain binding / negative regulation of glycogen catabolic process / positive regulation of nitric oxide mediated signal transduction / negative regulation of fatty acid metabolic process / Signaling by Insulin receptor / negative regulation of feeding behavior / IRS activation / Insulin processing / regulation of protein secretion / positive regulation of peptide hormone secretion / positive regulation of respiratory burst / amyloid-beta clearance / Regulation of gene expression in beta cells / negative regulation of acute inflammatory response / positive regulation of protein autophosphorylation / regulation of embryonic development / alpha-beta T cell activation / positive regulation of receptor internalization / protein kinase activator activity / insulin receptor substrate binding / positive regulation of dendritic spine maintenance / Synthesis, secretion, and deacylation of Ghrelin / negative regulation of respiratory burst involved in inflammatory response / epidermis development / negative regulation of protein secretion / negative regulation of gluconeogenesis / positive regulation of glycogen biosynthetic process / Signal attenuation / fatty acid homeostasis / phosphatidylinositol 3-kinase binding / FOXO-mediated transcription of oxidative stress, metabolic and neuronal genes / positive regulation of insulin receptor signaling pathway / negative regulation of lipid catabolic process / transport across blood-brain barrier / regulation of protein localization to plasma membrane / positive regulation of lipid biosynthetic process / heart morphogenesis / negative regulation of oxidative stress-induced intrinsic apoptotic signaling pathway / activation of protein kinase B activity / COPI-mediated anterograde transport / transport vesicle / nitric oxide-cGMP-mediated signaling / negative regulation of reactive oxygen species biosynthetic process / Insulin receptor recycling / insulin-like growth factor receptor binding / positive regulation of brown fat cell differentiation / dendrite membrane / NPAS4 regulates expression of target genes / neuron projection maintenance / endoplasmic reticulum-Golgi intermediate compartment membrane / positive regulation of MAP kinase activity / positive regulation of nitric-oxide synthase activity / positive regulation of mitotic nuclear division / Insulin receptor signalling cascade / receptor-mediated endocytosis / regulation of transmembrane transporter activity / positive regulation of glycolytic process / learning / positive regulation of long-term synaptic potentiation / positive regulation of cytokine production / endosome lumen / acute-phase response / positive regulation of D-glucose import / positive regulation of protein secretion / positive regulation of cell differentiation / Regulation of insulin secretion / insulin receptor binding / wound healing / placental growth factor receptor activity / insulin receptor activity / vascular endothelial growth factor receptor activity / hepatocyte growth factor receptor activity / macrophage colony-stimulating factor receptor activity / platelet-derived growth factor alpha-receptor activity / platelet-derived growth factor beta-receptor activity / stem cell factor receptor activity / boss receptor activity / protein tyrosine kinase collagen receptor activity / brain-derived neurotrophic factor receptor activity / transmembrane-ephrin receptor activity / GPI-linked ephrin receptor activity / epidermal growth factor receptor activity / fibroblast growth factor receptor activity / insulin-like growth factor receptor activity / receptor protein-tyrosine kinase
Similarity search - Function
Insulin receptor, trans-membrane domain / Insulin receptor trans-membrane segment / Tyrosine-protein kinase, insulin-like receptor / Tyrosine-protein kinase, receptor class II, conserved site / Receptor tyrosine kinase class II signature. / Insulin / Insulin family / Insulin-like / Insulin/IGF/Relaxin family / Insulin / insulin-like growth factor / relaxin family. ...Insulin receptor, trans-membrane domain / Insulin receptor trans-membrane segment / Tyrosine-protein kinase, insulin-like receptor / Tyrosine-protein kinase, receptor class II, conserved site / Receptor tyrosine kinase class II signature. / Insulin / Insulin family / Insulin-like / Insulin/IGF/Relaxin family / Insulin / insulin-like growth factor / relaxin family. / Insulin, conserved site / Insulin family signature. / Insulin-like superfamily / Receptor L-domain / Furin-like cysteine-rich domain / Receptor L-domain superfamily / Furin-like cysteine rich region / Receptor L domain / Furin-like repeat / Furin-like repeats / Growth factor receptor cysteine-rich domain superfamily / Fibronectin type III domain / : / Fibronectin type 3 domain / Fibronectin type-III domain profile. / Fibronectin type III / Fibronectin type III superfamily / Tyrosine-protein kinase, catalytic domain / Tyrosine kinase, catalytic domain / Tyrosine protein kinases specific active-site signature. / Tyrosine-protein kinase, active site / Serine-threonine/tyrosine-protein kinase, catalytic domain / Protein tyrosine and serine/threonine kinase / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Immunoglobulin-like fold / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
Insulin / Insulin receptor
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.3 Å
AuthorsNielsen, J.A. / Slaaby, R. / Boesen, T. / Hummelshoj, T. / Brandt, J. / Schluckebier, G. / Nissen, P.
Funding support Denmark, 1items
OrganizationGrant numberCountry
Other government Denmark
CitationJournal: J Mol Biol / Year: 2022
Title: Structural Investigations of Full-Length Insulin Receptor Dynamics and Signalling.
Authors: Jeppe Nielsen / Jakob Brandt / Thomas Boesen / Tina Hummelshøj / Rita Slaaby / Gerd Schluckebier / Poul Nissen /
Abstract: Insulin regulates glucose homeostasis via binding and activation of the insulin receptor dimer at two distinct pairs of binding sites 1 and 2. Here, we present cryo-EM studies of full-length human ...Insulin regulates glucose homeostasis via binding and activation of the insulin receptor dimer at two distinct pairs of binding sites 1 and 2. Here, we present cryo-EM studies of full-length human insulin receptor (hIR) in an active state obtained at non-saturating, physiologically relevant insulin conditions. Insulin binds asymmetrically to the receptor under these conditions, occupying up to three of the four possible binding sites. Deletion analysis of the receptor together with site specific peptides and insulin analogs used in binding studies show that both sites 1 and 2 are required for high insulin affinity. We identify a homotypic interaction of the fibronectin type III domain (FnIII-3) of IR resulting in tight interaction of membrane proximal domains of the active, asymmetric receptor dimer. Our results show how insulin binding at two distinct types of sites disrupts the autoinhibited apo-IR dimer and stabilizes the active dimer. We propose an insulin binding and activation mechanism, which is sequential, exhibits negative cooperativity, and is based on asymmetry at physiological insulin concentrations with one to three insulin molecules activating IR.
History
DepositionAug 12, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 2, 2022Provider: repository / Type: Initial release
Revision 1.1Feb 16, 2022Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.title
Revision 1.2Nov 20, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update

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Structure visualization

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Assembly

Deposited unit
A: Isoform Short of Insulin receptor
B: Isoform Short of Insulin receptor
C: Insulin
D: Insulin
E: Insulin
F: Insulin
I: Insulin
J: Insulin


Theoretical massNumber of molelcules
Total (without water)330,8488
Polymers330,8488
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area15010 Å2
ΔGint-91 kcal/mol
Surface area78760 Å2
MethodPISA

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Components

#1: Protein Isoform Short of Insulin receptor / IR


Mass: 156697.578 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: INSR / Production host: Cricetulus griseus (Chinese hamster)
References: UniProt: P06213, receptor protein-tyrosine kinase
#2: Protein/peptide Insulin


Mass: 2383.698 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: INS / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P01308
#3: Protein/peptide Insulin


Mass: 3433.953 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: INS / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P01308
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: DDM solubilised full-length human insulin receptor with three insulins bound
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.46 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Cricetulus griseus (Chinese hamster)
Buffer solutionpH: 7.8
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMSodium ChlorideNaCl1
250 mMHepesC8H18N2O4S1
30.03 w/v%DDMC24H46O111
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: C-flat-2/2
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 297 K / Details: Blotted for 3s prior to plunging

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm
Image recordingAverage exposure time: 15 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategoryDetails
7NAMDmodel fitting
13PHENIX1.19.2model refinementReal-space refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 7.3 Å / Resolution method: OTHER / Num. of particles: 32694 / Details: Masked FSC calculated with GSFSC in cryoSPARC2. / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT

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