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- PDB-6hn5: Leucine-zippered human insulin receptor ectodomain with single bo... -

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Basic information

Entry
Database: PDB / ID: 6hn5
TitleLeucine-zippered human insulin receptor ectodomain with single bound insulin - "upper" membrane-distal part
Components
  • (Insulin) x 2
  • Insulin receptor,General control protein GCN4
KeywordsSIGNALING PROTEIN / insulin / insulin receptor ectodomain / signal transduction
Function / homology
Function and homology information


regulation of female gonad development / positive regulation of meiotic cell cycle / protein localization to nuclear periphery / Activation of the AP-1 family of transcription factors / positive regulation of developmental growth / response to amino acid starvation / insulin-like growth factor II binding / mediator complex binding / negative regulation of ribosomal protein gene transcription by RNA polymerase II / positive regulation of cellular response to amino acid starvation ...regulation of female gonad development / positive regulation of meiotic cell cycle / protein localization to nuclear periphery / Activation of the AP-1 family of transcription factors / positive regulation of developmental growth / response to amino acid starvation / insulin-like growth factor II binding / mediator complex binding / negative regulation of ribosomal protein gene transcription by RNA polymerase II / positive regulation of cellular response to amino acid starvation / male sex determination / exocrine pancreas development / insulin receptor complex / insulin-like growth factor I binding / insulin receptor activity / nitrogen catabolite activation of transcription from RNA polymerase II promoter / 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 / TFIID-class transcription factor complex binding / 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 / amino acid biosynthetic process / negative regulation of acute inflammatory response / negative regulation of respiratory burst involved in inflammatory response / insulin receptor substrate binding / negative regulation of protein secretion / positive regulation of transcription initiation by RNA polymerase II / positive regulation of RNA polymerase II transcription preinitiation complex assembly / 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 / negative regulation of reactive oxygen species biosynthetic process / positive regulation of insulin receptor signaling pathway / transport vesicle / positive regulation of protein autophosphorylation / dendrite membrane / Insulin receptor recycling / insulin-like growth factor receptor binding / cellular response to amino acid starvation / 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
Similarity search - Function
Insulin receptor, trans-membrane domain / Insulin receptor trans-membrane segment / Tyrosine-protein kinase, insulin-like receptor / Basic region leucine zipper / Basic-leucine zipper (bZIP) domain signature. / Basic-leucine zipper (bZIP) domain profile. / basic region leucin zipper / Basic-leucine zipper domain superfamily / Basic-leucine zipper domain / Tyrosine-protein kinase, receptor class II, conserved site ...Insulin receptor, trans-membrane domain / Insulin receptor trans-membrane segment / Tyrosine-protein kinase, insulin-like receptor / Basic region leucine zipper / Basic-leucine zipper (bZIP) domain signature. / Basic-leucine zipper (bZIP) domain profile. / basic region leucin zipper / Basic-leucine zipper domain superfamily / Basic-leucine zipper domain / 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 / 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: 3.2 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 11, 2019Group: Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB
Revision 2.0Jul 29, 2020Group: Atomic model / Data collection ...Atomic model / Data collection / Derived calculations / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / entity / pdbx_branch_scheme / pdbx_chem_comp_identifier / pdbx_entity_branch / pdbx_entity_branch_descriptor / pdbx_entity_branch_link / pdbx_entity_branch_list / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / pdbx_struct_assembly_gen / struct_asym / struct_conn / struct_site / struct_site_gen
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_asym_id / _atom_site.auth_seq_id / _atom_site.label_asym_id / _atom_site.label_entity_id / _chem_comp.name / _chem_comp.type / _pdbx_entity_nonpoly.entity_id / _pdbx_entity_nonpoly.name / _pdbx_struct_assembly_gen.asym_id_list / _struct_conn.pdbx_role / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 2.1Mar 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

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Structure visualization

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  • Deposited structure unit
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  • EMDB-0247
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Assembly

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


Theoretical massNumber of molelcules
Total (without water)221,68914
Polymers219,2744
Non-polymers2,41510
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area9890 Å2
ΔGint7 kcal/mol
Surface area46790 Å2
MethodPISA

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Components

#1: Protein/peptide Insulin /


Mass: 2383.698 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) / References: UniProt: P01308
#2: Protein/peptide Insulin /


Mass: 3433.953 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) / References: UniProt: P01308
#3: Protein 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
#4: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#5: 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 / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 9
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

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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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Leucine zippered human insulin receptor ectodomain (IR-A isoform, "deltabeta" mutant) in complex with insulin and two Fv 83-7 modules : "upper" membrane-distal partCOMPLEXNote: 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.#1-#30MULTIPLE SOURCES
2insulinCOMPLEX#1-#21RECOMBINANT
3Leucine zippered human insulin receptor ectodomainCOMPLEX#31RECOMBINANT
Molecular weightUnits: MEGADALTONS / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Cricetulus griseus (Chinese hamster)10029
33synthetic construct (others)32630
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
Specimen supportDetails: PELCO easiGlow factory settings / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-2/2
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 FIELDBright-field microscopy / 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
10RELION2.1initial Euler assignment
11RELION2.1final Euler assignment
12RELION2.1classification
13RELION2.13D reconstruction
20PHENIX1.13-2998-1692model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 747074
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 213867 / Algorithm: BACK PROJECTION / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Atomic model buildingPDB-ID: 4ZXB

4zxb

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