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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 / positive regulation of developmental growth / insulin-like growth factor II binding / male sex determination / exocrine pancreas development / insulin receptor complex / insulin-like growth factor I binding / insulin receptor activity / positive regulation of protein-containing complex disassembly ...regulation of female gonad development / positive regulation of meiotic cell cycle / positive regulation of developmental growth / insulin-like growth factor II binding / male sex determination / exocrine pancreas development / insulin receptor complex / insulin-like growth factor I binding / insulin receptor activity / positive regulation of protein-containing complex disassembly / cargo receptor activity / dendritic spine maintenance / insulin binding / PTB domain binding / negative regulation of NAD(P)H oxidase activity / neuronal cell body membrane / adrenal gland development / negative regulation of glycogen catabolic process / regulation of cellular amino acid metabolic process / Signaling by Insulin receptor / IRS activation / nitric oxide-cGMP-mediated signaling / negative regulation of fatty acid metabolic process / Insulin processing / negative regulation of feeding behavior / regulation of protein secretion / amyloid-beta clearance / positive regulation of peptide hormone secretion / activation of protein kinase activity / Regulation of gene expression in beta cells / positive regulation of respiratory burst / regulation of embryonic development / positive regulation of receptor internalization / transport across blood-brain barrier / positive regulation of dendritic spine maintenance / alpha-beta T cell activation / negative regulation of acute inflammatory response / negative regulation of respiratory burst involved in inflammatory response / insulin receptor substrate binding / negative regulation of protein secretion / fatty acid homeostasis / Synthesis, secretion, and deacylation of Ghrelin / epidermis development / positive regulation of glycogen biosynthetic process / positive regulation of lipid biosynthetic process / Signal attenuation / FOXO-mediated transcription of oxidative stress, metabolic and neuronal genes / negative regulation of gluconeogenesis / positive regulation of nitric oxide mediated signal transduction / regulation of protein localization to plasma membrane / COPI-mediated anterograde transport / phosphatidylinositol 3-kinase binding / negative regulation of lipid catabolic process / heart morphogenesis / negative regulation of oxidative stress-induced intrinsic apoptotic signaling pathway / positive regulation of insulin receptor signaling pathway / negative regulation of reactive oxygen species biosynthetic process / transport vesicle / positive regulation of protein autophosphorylation / dendrite membrane / Insulin receptor recycling / insulin-like growth factor receptor binding / receptor-mediated endocytosis / NPAS4 regulates expression of target genes / positive regulation of protein metabolic process / neuron projection maintenance / endoplasmic reticulum-Golgi intermediate compartment membrane / positive regulation of brown fat cell differentiation / activation of protein kinase B activity / positive regulation of glycolytic process / Insulin receptor signalling cascade / positive regulation of mitotic nuclear division / learning / Regulation of insulin secretion / positive regulation of nitric-oxide synthase activity / positive regulation of long-term synaptic potentiation / caveola / endosome lumen / positive regulation of cytokine production / acute-phase response / positive regulation of protein secretion / regulation of transmembrane transporter activity / positive regulation of cell differentiation / positive regulation of glucose import / negative regulation of proteolysis / regulation of synaptic plasticity / positive regulation of MAP kinase activity / wound healing / insulin receptor binding / negative regulation of protein catabolic process / positive regulation of neuron projection development / hormone activity / receptor internalization / receptor protein-tyrosine kinase / memory / cellular response to growth factor stimulus / cognition / Golgi lumen / vasodilation / positive regulation of protein localization to nucleus
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/IGF/Relaxin family / Insulin, conserved site / Insulin family signature. ...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/IGF/Relaxin family / Insulin, conserved site / Insulin family signature. / Insulin-like / Insulin / insulin-like growth factor / relaxin family. / 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 / Protein tyrosine and serine/threonine kinase / Serine-threonine/tyrosine-protein kinase, catalytic domain / 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

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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
Water0
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

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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 ChlorideNaClSodium chloride1
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 FIELDBright-field microscopy / 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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