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- PDB-6ni2: Stabilized beta-arrestin 1-V2T subcomplex of a GPCR-G protein-bet... -

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Basic information

Entry
Database: PDB / ID: 6ni2
TitleStabilized beta-arrestin 1-V2T subcomplex of a GPCR-G protein-beta-arrestin mega-complex
Components
  • Beta-arrestin-1
  • Fab30 Heavy Chain
  • Fab30 Light Chain
  • Nanobody 32
  • Vasopressin V2 receptor
KeywordsSIGNALING PROTEIN/IMMUNE SYSTEM / arrestin / GPCR signaling transducer / scaffolding protein / SIGNALING PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


TGFBR3 regulates TGF-beta signaling / MAP2K and MAPK activation / Activation of SMO / renal water retention / Golgi Associated Vesicle Biogenesis / Defective AVP does not bind AVPR2 and causes neurohypophyseal diabetes insipidus (NDI) / Vasopressin-like receptors / regulation of systemic arterial blood pressure by vasopressin / vasopressin receptor activity / Lysosome Vesicle Biogenesis ...TGFBR3 regulates TGF-beta signaling / MAP2K and MAPK activation / Activation of SMO / renal water retention / Golgi Associated Vesicle Biogenesis / Defective AVP does not bind AVPR2 and causes neurohypophyseal diabetes insipidus (NDI) / Vasopressin-like receptors / regulation of systemic arterial blood pressure by vasopressin / vasopressin receptor activity / Lysosome Vesicle Biogenesis / AP-2 adaptor complex binding / Ub-specific processing proteases / clathrin coat of coated pit / clathrin heavy chain binding / Cargo recognition for clathrin-mediated endocytosis / hemostasis / desensitization of G protein-coupled receptor signaling pathway / telencephalon development / Clathrin-mediated endocytosis / clathrin-dependent endocytosis / acetylcholine receptor binding / G protein-coupled receptor internalization / inositol hexakisphosphate binding / Thrombin signalling through proteinase activated receptors (PARs) / G alpha (s) signalling events / clathrin binding / small molecule binding / positive regulation of vasoconstriction / pseudopodium / positive regulation of systemic arterial blood pressure / phosphatidylinositol-3,4,5-trisphosphate binding / positive regulation of intracellular signal transduction / positive regulation of receptor internalization / endocytic vesicle / negative regulation of Notch signaling pathway / activation of adenylate cyclase activity / cellular response to hormone stimulus / response to cytokine / clathrin-coated endocytic vesicle membrane / G protein-coupled receptor binding / receptor internalization / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / positive regulation of protein phosphorylation / Vasopressin regulates renal water homeostasis via Aquaporins / Cargo recognition for clathrin-mediated endocytosis / protein transport / Clathrin-mediated endocytosis / cytoplasmic vesicle / ubiquitin-dependent protein catabolic process / G alpha (s) signalling events / molecular adaptor activity / positive regulation of ERK1 and ERK2 cascade / endosome / G protein-coupled receptor signaling pathway / negative regulation of cell population proliferation / positive regulation of cell population proliferation / positive regulation of gene expression / perinuclear region of cytoplasm / endoplasmic reticulum / Golgi apparatus / signal transduction / nucleus / membrane / plasma membrane / cytoplasm / cytosol
Similarity search - Function
Vasopressin V2 receptor / Vasopressin receptor / Arrestin, conserved site / Arrestins signature. / Arrestin / Arrestin, N-terminal / Arrestin-like, N-terminal / Arrestin C-terminal-like domain / Arrestin (or S-antigen), N-terminal domain / Arrestin (or S-antigen), C-terminal domain ...Vasopressin V2 receptor / Vasopressin receptor / Arrestin, conserved site / Arrestins signature. / Arrestin / Arrestin, N-terminal / Arrestin-like, N-terminal / Arrestin C-terminal-like domain / Arrestin (or S-antigen), N-terminal domain / Arrestin (or S-antigen), C-terminal domain / Arrestin (or S-antigen), C-terminal domain / Arrestin-like, C-terminal / G-protein coupled receptors family 1 signature. / G protein-coupled receptor, rhodopsin-like / GPCR, rhodopsin-like, 7TM / G-protein coupled receptors family 1 profile. / 7 transmembrane receptor (rhodopsin family) / Immunoglobulin E-set
Similarity search - Domain/homology
Beta-arrestin-1 / Vasopressin V2 receptor
Similarity search - Component
Biological speciesLama glama (llama)
Bos taurus (domestic cattle)
Homo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsNguyen, A.H. / Thomsen, A.R.B. / Cahill, T.J. / des Georges, A. / Lefkowitz, R.J.
Funding support United States, 2items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)2R01-HL016037-47 United States
CitationJournal: Nat Struct Mol Biol / Year: 2019
Title: Structure of an endosomal signaling GPCR-G protein-β-arrestin megacomplex.
Authors: Anthony H Nguyen / Alex R B Thomsen / Thomas J Cahill / Rick Huang / Li-Yin Huang / Tara Marcink / Oliver B Clarke / Søren Heissel / Ali Masoudi / Danya Ben-Hail / Fadi Samaan / Venkata P ...Authors: Anthony H Nguyen / Alex R B Thomsen / Thomas J Cahill / Rick Huang / Li-Yin Huang / Tara Marcink / Oliver B Clarke / Søren Heissel / Ali Masoudi / Danya Ben-Hail / Fadi Samaan / Venkata P Dandey / Yong Zi Tan / Chuan Hong / Jacob P Mahoney / Sarah Triest / John Little / Xin Chen / Roger Sunahara / Jan Steyaert / Henrik Molina / Zhiheng Yu / Amedee des Georges / Robert J Lefkowitz /
Abstract: Classically, G-protein-coupled receptors (GPCRs) are thought to activate G protein from the plasma membrane and are subsequently desensitized by β-arrestin (β-arr). However, some GPCRs continue to ...Classically, G-protein-coupled receptors (GPCRs) are thought to activate G protein from the plasma membrane and are subsequently desensitized by β-arrestin (β-arr). However, some GPCRs continue to signal through G protein from internalized compartments, mediated by a GPCR-G protein-β-arr 'megaplex'. Nevertheless, the molecular architecture of the megaplex remains unknown. Here, we present its cryo-electron microscopy structure, which shows simultaneous engagement of human G protein and bovine β-arr to the core and phosphorylated tail, respectively, of a single active human chimeric β-adrenergic receptor with the C-terminal tail of the arginine vasopressin type 2 receptor (βVR). All three components adopt their canonical active conformations, suggesting that a single megaplex GPCR is capable of simultaneously activating G protein and β-arr. Our findings provide a structural basis for GPCR-mediated sustained internalized G protein signaling.
History
DepositionDec 26, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 20, 2019Provider: repository / Type: Initial release
Revision 1.1Dec 4, 2019Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.2Dec 18, 2019Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Nov 20, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

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Assembly

Deposited unit
A: Nanobody 32
B: Beta-arrestin-1
H: Fab30 Heavy Chain
L: Fab30 Light Chain
V: Vasopressin V2 receptor


Theoretical massNumber of molelcules
Total (without water)110,0385
Polymers110,0385
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: immunoprecipitation, gel filtration, microscopy, fluorescence resonance energy transfer
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area6340 Å2
ΔGint-49 kcal/mol
Surface area33950 Å2

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Components

#1: Antibody Nanobody 32


Mass: 13665.136 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)
#2: Protein Beta-arrestin-1 / Arrestin beta-1 / Arrestin-2


Mass: 44309.578 Da / Num. of mol.: 1 / Fragment: UNP residues 6-361
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bos taurus (domestic cattle) / Gene: ARRB1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P17870
#3: Antibody Fab30 Heavy Chain


Mass: 25512.354 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: Antibody Fab30 Light Chain


Mass: 23435.064 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: Protein/peptide Vasopressin V2 receptor / V2R / AVPR V2 / Antidiuretic hormone receptor / Renal-type arginine vasopressin receptor


Mass: 3115.718 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: AVPR2, ADHR, DIR, DIR3, V2R / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P30518
Has ligand of interestY
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

Component
IDNameTypeEntity IDParent-IDSource
1Stabilized beta-arrestin 1-V2T subcomplex of a GPCR-G protein-beta-arrestin mega-complexCOMPLEXall0MULTIPLE SOURCES
2Nanobody 32COMPLEX#11RECOMBINANT
3Beta-arrestin-1COMPLEX#21RECOMBINANT
4Fab30COMPLEX#3-#41NATURAL
5Vasopressin 2 TailCOMPLEX#51RECOMBINANT
Molecular weightUnits: MEGADALTONS / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Lama glama (llama)9844
23Bos taurus (domestic cattle)9913
34synthetic construct (others)32630
45Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli (E. coli)562
23Spodoptera frugiperda (fall armyworm)7108
45Spodoptera frugiperda (fall armyworm)7108
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unidentified
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
Image recordingElectron dose: 104 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.13_2998: / Classification: refinement
EM software
IDNameCategory
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 230021 / Symmetry type: POINT

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