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- PDB-8eyx: Cryo-EM structure of 4 insulins bound full-length mouse IR mutant... -

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Basic information

Entry
Database: PDB / ID: 8eyx
TitleCryo-EM structure of 4 insulins bound full-length mouse IR mutant with physically decoupled alpha CTs (C684S/C685S/C687S; denoted as IR-3CS) Asymmetric conformation 1
Components
  • Insulin
  • Insulin receptor
KeywordsSIGNALING PROTEIN / Insulin receptor / insulin
Function / homology
Function and homology information


3-phosphoinositide-dependent protein kinase binding / Signaling by Insulin receptor / yolk / IRS activation / Insulin receptor signalling cascade / Signal attenuation / Insulin receptor recycling / lipoic acid binding / PI5P, PP2A and IER3 Regulate PI3K/AKT Signaling / positive regulation of glycoprotein biosynthetic process ...3-phosphoinositide-dependent protein kinase binding / Signaling by Insulin receptor / yolk / IRS activation / Insulin receptor signalling cascade / Signal attenuation / Insulin receptor recycling / lipoic acid binding / PI5P, PP2A and IER3 Regulate PI3K/AKT Signaling / positive regulation of glycoprotein biosynthetic process / regulation of female gonad development / regulation of hydrogen peroxide metabolic process / positive regulation of meiotic cell cycle / insulin-like growth factor II binding / positive regulation of developmental growth / male sex determination / nuclear lumen / insulin receptor complex / insulin-like growth factor I binding / insulin receptor activity / positive regulation of protein-containing complex disassembly / exocrine pancreas development / dendritic spine maintenance / adrenal gland development / insulin binding / cargo receptor activity / negative regulation of glycogen catabolic process / : / negative regulation of fatty acid metabolic process / PTB domain binding / Signaling by Insulin receptor / negative regulation of feeding behavior / IRS activation / Insulin processing / regulation of protein secretion / positive regulation of peptide hormone secretion / neuronal cell body membrane / negative regulation of acute inflammatory response / Regulation of gene expression in beta cells / positive regulation of respiratory burst / alpha-beta T cell activation / amyloid-beta clearance / regulation of embryonic development / insulin receptor substrate binding / positive regulation of receptor internalization / response to tumor necrosis factor / Synthesis, secretion, and deacylation of Ghrelin / epidermis development / positive regulation of phosphorylation / negative regulation of protein secretion / positive regulation of dendritic spine maintenance / negative regulation of gluconeogenesis / fatty acid homeostasis / positive regulation of glycogen biosynthetic process / positive regulation of insulin receptor signaling pathway / Signal attenuation / FOXO-mediated transcription of oxidative stress, metabolic and neuronal genes / protein kinase activator activity / negative regulation of lipid catabolic process / negative regulation of respiratory burst involved in inflammatory response / positive regulation of lipid biosynthetic process / negative regulation of oxidative stress-induced intrinsic apoptotic signaling pathway / nitric oxide-cGMP-mediated signaling / regulation of protein localization to plasma membrane / positive regulation of nitric-oxide synthase activity / heart morphogenesis / phosphatidylinositol 3-kinase binding / transport vesicle / Insulin receptor recycling / COPI-mediated anterograde transport / negative regulation of reactive oxygen species biosynthetic process / positive regulation of brown fat cell differentiation / insulin-like growth factor receptor binding / NPAS4 regulates expression of target genes / neuron projection maintenance / positive regulation of mitotic nuclear division / endoplasmic reticulum-Golgi intermediate compartment membrane / Insulin receptor signalling cascade / receptor-mediated endocytosis / dendrite membrane / positive regulation of glycolytic process / animal organ morphogenesis / endosome lumen / positive regulation of cytokine production / acute-phase response / positive regulation of D-glucose import across plasma membrane / insulin receptor binding / positive regulation of long-term synaptic potentiation / positive regulation of protein secretion / positive regulation of cell differentiation / wound healing / Regulation of insulin secretion / hormone activity / receptor protein-tyrosine kinase / positive regulation of neuron projection development / negative regulation of protein catabolic process / response to nutrient levels / caveola / regulation of synaptic plasticity / 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-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 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 speciesMus musculus (house mouse)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsLi, J. / Wu, J.Y. / Hall, C. / Bai, X.C. / Choi, E.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM136976 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM142937 United States
CitationJournal: Elife / Year: 2022
Title: Molecular basis for the role of disulfide-linked αCTs in the activation of insulin-like growth factor 1 receptor and insulin receptor.
Authors: Jie Li / Jiayi Wu / Catherine Hall / Xiao-Chen Bai / Eunhee Choi /
Abstract: The insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R) control metabolic homeostasis and cell growth and proliferation. The IR and IGF1R form similar disulfide bonds linked ...The insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R) control metabolic homeostasis and cell growth and proliferation. The IR and IGF1R form similar disulfide bonds linked homodimers in the apo-state; however, their ligand binding properties and the structures in the active state differ substantially. It has been proposed that the disulfide-linked C-terminal segment of α-chain (αCTs) of the IR and IGF1R control the cooperativity of ligand binding and regulate the receptor activation. Nevertheless, the molecular basis for the roles of disulfide-linked αCTs in IR and IGF1R activation are still unclear. Here, we report the cryo-EM structures of full-length mouse IGF1R/IGF1 and IR/insulin complexes with modified αCTs that have increased flexibility. Unlike the -shaped asymmetric IGF1R dimer with a single IGF1 bound, the IGF1R with the enhanced flexibility of αCTs can form a -shaped symmetric dimer with two IGF1s bound. Meanwhile, the IR with non-covalently linked αCTs predominantly adopts an asymmetric conformation with four insulins bound, which is distinct from the -shaped symmetric IR. Using cell-based experiments, we further showed that both IGF1R and IR with the modified αCTs cannot activate the downstream signaling potently. Collectively, our studies demonstrate that the certain structural rigidity of disulfide-linked αCTs is critical for optimal IR and IGF1R signaling activation.
History
DepositionOct 29, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 9, 2022Provider: repository / Type: Initial release
Revision 1.1Dec 7, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Oct 23, 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

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Insulin receptor
B: Insulin receptor
D: Insulin
E: Insulin
F: Insulin
G: Insulin


Theoretical massNumber of molelcules
Total (without water)354,3286
Polymers354,3286
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Insulin receptor / IR


Mass: 153184.406 Da / Num. of mol.: 2 / Mutation: C684S,C685S,C687S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Insr / Production host: Homo sapiens (human)
References: UniProt: P15208, receptor protein-tyrosine kinase
#2: Protein
Insulin


Mass: 11989.862 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human)
Description: purchased from Sigma-Aldrich, expressed in yeast (proprietary host)
Gene: INS / Production host: Saccharomyces cerevisiae / 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: Cryo-EM structure of 4 insulins bound full-length mouse IR mutant with physically decoupled alpha CTs (C684S/C685S/C687S; denoted as IR-3CS) Asymmetric conformation 1
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4
SpecimenConc.: 6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE

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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: 2600 nm / Nominal defocus min: 1600 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameCategory
1RELIONparticle selection
2SerialEMimage acquisition
4GctfCTF correction
7Cootmodel fitting
12RELION3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3283617
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 101391 / Symmetry type: POINT

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