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- PDB-6hn4: Leucine-zippered human insulin receptor ectodomain with single bo... -

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Basic information

Entry
Database: PDB / ID: 6hn4
TitleLeucine-zippered human insulin receptor ectodomain with single bound insulin - "lower" membrane-proximal part
ComponentsInsulin receptor,Insulin receptor,General control protein GCN4
KeywordsSIGNALING PROTEIN / insulin / insulin receptor ectodomain / signal transdution
Function / homology
Function and homology information


FCERI mediated MAPK activation / protein localization to nuclear periphery / regulation of female gonad development / Activation of the AP-1 family of transcription factors / positive regulation of meiotic cell cycle / response to amino acid starvation / negative regulation of ribosomal protein gene transcription by RNA polymerase II / positive regulation of cellular response to amino acid starvation / mediator complex binding / insulin-like growth factor II binding ...FCERI mediated MAPK activation / protein localization to nuclear periphery / regulation of female gonad development / Activation of the AP-1 family of transcription factors / positive regulation of meiotic cell cycle / response to amino acid starvation / negative regulation of ribosomal protein gene transcription by RNA polymerase II / positive regulation of cellular response to amino acid starvation / mediator complex binding / insulin-like growth factor II binding / positive regulation of developmental growth / male sex determination / insulin receptor complex / insulin-like growth factor I binding / positive regulation of protein-containing complex disassembly / insulin receptor activity / Oxidative Stress Induced Senescence / exocrine pancreas development / dendritic spine maintenance / cargo receptor activity / insulin binding / adrenal gland development / neuronal cell body membrane / PTB domain binding / Signaling by Insulin receptor / IRS activation / positive regulation of respiratory burst / amyloid-beta clearance / TFIID-class transcription factor complex binding / amino acid biosynthetic process / positive regulation of receptor internalization / regulation of embryonic development / insulin receptor substrate binding / positive regulation of RNA polymerase II transcription preinitiation complex assembly / protein kinase activator activity / epidermis development / positive regulation of glycogen biosynthetic process / positive regulation of transcription initiation by RNA polymerase II / Signal attenuation / heart morphogenesis / cellular response to nutrient levels / transport across blood-brain barrier / phosphatidylinositol 3-kinase binding / Insulin receptor recycling / insulin-like growth factor receptor binding / dendrite membrane / neuron projection maintenance / positive regulation of mitotic nuclear division / Insulin receptor signalling cascade / receptor-mediated endocytosis / cellular response to amino acid starvation / positive regulation of glycolytic process / positive regulation of D-glucose import / learning / receptor protein-tyrosine kinase / caveola / cellular response to growth factor stimulus / receptor internalization / RNA polymerase II transcription regulator complex / memory / male gonad development / cellular response to insulin stimulus / positive regulation of nitric oxide biosynthetic process / insulin receptor signaling pathway / late endosome / glucose homeostasis / amyloid-beta binding / protein autophosphorylation / PI5P, PP2A and IER3 Regulate PI3K/AKT Signaling / DNA-binding transcription activator activity, RNA polymerase II-specific / protein tyrosine kinase activity / transcription regulator complex / sequence-specific DNA binding / RNA polymerase II-specific DNA-binding transcription factor binding / DNA-binding transcription factor activity, RNA polymerase II-specific / lysosome / positive regulation of canonical NF-kappaB signal transduction / positive regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / receptor complex / endosome membrane / positive regulation of MAPK cascade / intracellular signal transduction / positive regulation of cell migration / RNA polymerase II cis-regulatory region sequence-specific DNA binding / G protein-coupled receptor signaling pathway / DNA-binding transcription factor activity / protein domain specific binding / axon / external side of plasma membrane / positive regulation of cell population proliferation / chromatin binding / regulation of DNA-templated transcription / symbiont entry into host cell / GTP binding / protein-containing complex binding / positive regulation of DNA-templated transcription / negative regulation of transcription by RNA polymerase II / positive regulation of transcription by RNA polymerase II / extracellular exosome / ATP binding
Similarity search - Function
Insulin receptor, trans-membrane domain / Insulin receptor trans-membrane segment / : / Basic region leucine zipper / Tyrosine-protein kinase, insulin-like receptor / Basic-leucine zipper (bZIP) domain signature. / Basic-leucine zipper (bZIP) domain profile. / basic region leucin zipper / Basic-leucine zipper domain / Basic-leucine zipper domain superfamily ...Insulin receptor, trans-membrane domain / Insulin receptor trans-membrane segment / : / Basic region leucine zipper / Tyrosine-protein kinase, insulin-like receptor / Basic-leucine zipper (bZIP) domain signature. / Basic-leucine zipper (bZIP) domain profile. / basic region leucin zipper / Basic-leucine zipper domain / Basic-leucine zipper domain superfamily / Tyrosine-protein kinase, receptor class II, conserved site / Receptor tyrosine kinase class II signature. / 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 / Fibronectin type III domain / 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 / 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
General control transcription factor GCN4 / Insulin receptor
Similarity search - Component
Biological speciesHomo sapiens (human)
Saccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsWeis, F. / Menting, J.G. / Margetts, M.B. / Chan, S.J. / Xu, Y. / Tennagels, N. / Wohlfart, P. / Langer, T. / Mueller, C.W. / Dreyer, M.K. / Lawrence, M.C.
Funding support Australia, United States, 4items
OrganizationGrant numberCountry
National Health and Medical Research Council (Australia)APP1099595 Australia
National Health and Medical Research Council (Australia)APP1128553 Australia
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)DK013914 United States
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)UC DRTC DK020595 United States
CitationJournal: Nat Commun / Year: 2018
Title: The signalling conformation of the insulin receptor ectodomain.
Authors: Felix Weis / John G Menting / Mai B Margetts / Shu Jin Chan / Yibin Xu / Norbert Tennagels / Paulus Wohlfart / Thomas Langer / Christoph W Müller / Matthias K Dreyer / Michael C Lawrence /
Abstract: Understanding the structural biology of the insulin receptor and how it signals is of key importance in the development of insulin analogs to treat diabetes. We report here a cryo-electron microscopy ...Understanding the structural biology of the insulin receptor and how it signals is of key importance in the development of insulin analogs to treat diabetes. We report here a cryo-electron microscopy structure of a single insulin bound to a physiologically relevant, high-affinity version of the receptor ectodomain, the latter generated through attachment of C-terminal leucine zipper elements to overcome the conformational flexibility associated with ectodomain truncation. The resolution of the cryo-electron microscopy maps is 3.2 Å in the insulin-binding region and 4.2 Å in the membrane-proximal region. The structure reveals how the membrane proximal domains of the receptor come together to effect signalling and how insulin's negative cooperativity of binding likely arises. Our structure further provides insight into the high affinity of certain super-mitogenic insulins. Together, these findings provide a new platform for insulin analog investigation and design.
History
DepositionSep 14, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 21, 2018Provider: repository / Type: Initial release
Revision 1.1Nov 6, 2019Group: Data collection / Refinement description / Category: em_3d_fitting / Item: _em_3d_fitting.target_criteria
Revision 1.2Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]
Revision 1.3Jul 29, 2020Group: Data collection / Derived calculations / Structure summary
Category: chem_comp / entity ...chem_comp / entity / pdbx_chem_comp_identifier / pdbx_entity_nonpoly / struct_conn / struct_site / struct_site_gen
Item: _chem_comp.name / _chem_comp.type ..._chem_comp.name / _chem_comp.type / _entity.pdbx_description / _pdbx_entity_nonpoly.name / _struct_conn.pdbx_role
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 1.4Mar 30, 2022Group: Author supporting evidence / Database references / Structure summary
Category: chem_comp / database_2 / pdbx_audit_support
Item: _chem_comp.pdbx_synonyms / _database_2.pdbx_DOI ..._chem_comp.pdbx_synonyms / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_audit_support.funding_organization
Revision 1.5Nov 13, 2024Group: Data collection / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update

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Structure visualization

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Assembly

Deposited unit
E: Insulin receptor,Insulin receptor,General control protein GCN4
F: Insulin receptor,Insulin receptor,General control protein GCN4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)214,7848
Polymers213,4562
Non-polymers1,3276
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area4080 Å2
ΔGint24 kcal/mol
Surface area37960 Å2
MethodPISA

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Components

#1: Protein Insulin receptor,Insulin receptor,General control protein GCN4 / IR / IR / Amino acid biosynthesis regulatory protein


Mass: 106728.211 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Gene: INSR, GCN4, AAS3, ARG9, YEL009C / Plasmid: pEE14 / Strain: ATCC 204508 / S288c / Cell line (production host): Lec8 / Production host: Cricetulus griseus (Chinese hamster)
References: UniProt: P06213, UniProt: P03069, receptor protein-tyrosine kinase
#2: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
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: Leucine zippered human insulin receptor ectodomain (IR-A isoform, "deltabeta" mutant) in complex with insulin and two Fv 83-7 modules : "lower" membrane-proximal part
Type: COMPLEX
Details: Note: Attached to the leucine-zippered insulin receptor ectodomain are two Fv 83-7 modules. One of these is present within this map volume but it is very poorly ordered and thus left ...Details: Note: Attached to the leucine-zippered insulin receptor ectodomain are two Fv 83-7 modules. One of these is present within this map volume but it is very poorly ordered and thus left completely unmodelled. See the manuscript for further details.
Entity ID: #1 / Source: RECOMBINANT
Molecular weightUnits: MEGADALTONS / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Cricetulus griseus (Chinese hamster) / Cell: Lec8 / Plasmid: pEE14
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMHEPES-NaOH1
20.02 %sodium azide1
SpecimenConc.: 0.094 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283.15 K

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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 magnification: 130000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 16 sec. / Electron dose: 1.85 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2287
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 20 / Used frames/image: 1-20

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Processing

SoftwareName: PHENIX / Version: 1.13-2998_1692: / Classification: refinement
EM software
IDNameVersionCategory
1RELION2.1particle selection
2SerialEMimage acquisition
4GctfCTF correction
7UCSF Chimera1.11.2model fitting
8Coot0.8.7model fitting
10PHENIX1.13-2998-1692model refinement
11RELION2.1initial Euler assignment
12RELION2.1final Euler assignment
13RELION2.1classification
14RELION2.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 747074
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 98481 / Algorithm: BACK PROJECTION / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL / Target criteria: Cross-correlation coefficient
Atomic model buildingPDB-ID: 4ZXB

4zxb


Accession code: 4ZXB / Source name: PDB / Type: experimental model

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