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- PDB-6ni3: B2V2R-Gs protein subcomplex of a GPCR-G protein-beta-arrestin meg... -

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Basic information

Entry
Database: PDB / ID: 6ni3
TitleB2V2R-Gs protein subcomplex of a GPCR-G protein-beta-arrestin mega-complex
Components
  • (Guanine nucleotide-binding protein ...) x 3
  • Endolysin,Beta-2 adrenergic receptor chimera
  • Nanobody 35
KeywordsSIGNALING PROTEIN/IMMUNE SYSTEM / GPCR / Gs protein / signaling transducer / SIGNALING PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


positive regulation of mini excitatory postsynaptic potential / beta2-adrenergic receptor activity / AMPA selective glutamate receptor signaling pathway / norepinephrine binding / positive regulation of autophagosome maturation / norepinephrine-epinephrine-mediated vasodilation involved in regulation of systemic arterial blood pressure / heat generation / Adrenoceptors / activation of transmembrane receptor protein tyrosine kinase activity / negative regulation of smooth muscle contraction ...positive regulation of mini excitatory postsynaptic potential / beta2-adrenergic receptor activity / AMPA selective glutamate receptor signaling pathway / norepinephrine binding / positive regulation of autophagosome maturation / norepinephrine-epinephrine-mediated vasodilation involved in regulation of systemic arterial blood pressure / heat generation / Adrenoceptors / activation of transmembrane receptor protein tyrosine kinase activity / negative regulation of smooth muscle contraction / positive regulation of lipophagy / negative regulation of G protein-coupled receptor signaling pathway / negative regulation of multicellular organism growth / adrenergic receptor signaling pathway / response to psychosocial stress / endosome to lysosome transport / diet induced thermogenesis / neuronal dense core vesicle / positive regulation of cAMP/PKA signal transduction / adenylate cyclase binding / PKA activation in glucagon signalling / smooth muscle contraction / hair follicle placode formation / developmental growth / D1 dopamine receptor binding / bone resorption / positive regulation of bone mineralization / potassium channel regulator activity / intracellular transport / vascular endothelial cell response to laminar fluid shear stress / brown fat cell differentiation / renal water homeostasis / intercellular bridge / Hedgehog 'off' state / viral release from host cell by cytolysis / regulation of sodium ion transport / adenylate cyclase-activating adrenergic receptor signaling pathway / activation of adenylate cyclase activity / peptidoglycan catabolic process / regulation of insulin secretion / cellular response to glucagon stimulus / receptor-mediated endocytosis / adenylate cyclase activator activity / response to cold / trans-Golgi network membrane / negative regulation of inflammatory response to antigenic stimulus / clathrin-coated endocytic vesicle membrane / bone development / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / G-protein beta/gamma-subunit complex binding / platelet aggregation / Olfactory Signaling Pathway / cognition / Activation of the phototransduction cascade / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / G protein-coupled acetylcholine receptor signaling pathway / adenylate cyclase-activating G protein-coupled receptor signaling pathway / G-protein activation / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / Prostacyclin signalling through prostacyclin receptor / G beta:gamma signalling through CDC42 / Glucagon signaling in metabolic regulation / cellular response to amyloid-beta / G beta:gamma signalling through BTK / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / ADP signalling through P2Y purinoceptor 12 / Sensory perception of sweet, bitter, and umami (glutamate) taste / photoreceptor disc membrane / Glucagon-type ligand receptors / Adrenaline,noradrenaline inhibits insulin secretion / cell wall macromolecule catabolic process / Vasopressin regulates renal water homeostasis via Aquaporins / mitotic spindle / G alpha (z) signalling events / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / cellular response to catecholamine stimulus / ADORA2B mediated anti-inflammatory cytokines production / ADP signalling through P2Y purinoceptor 1 / G beta:gamma signalling through PI3Kgamma / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / adenylate cyclase-activating dopamine receptor signaling pathway / sensory perception of smell / lysozyme / lysozyme activity / GPER1 signaling / Inactivation, recovery and regulation of the phototransduction cascade / cellular response to prostaglandin E stimulus / G-protein beta-subunit binding / heterotrimeric G-protein complex / G alpha (12/13) signalling events / Cargo recognition for clathrin-mediated endocytosis / sensory perception of taste / extracellular vesicle / signaling receptor complex adaptor activity / Thrombin signalling through proteinase activated receptors (PARs) / Clathrin-mediated endocytosis / amyloid-beta binding
Similarity search - Function
Transducin (heterotrimeric G protein), gamma chain / G Protein Gi Gamma 2 / Beta 2 adrenoceptor / Adrenoceptor family / G-protein alpha subunit, group S / Endolysin T4 type / T4-type lysozyme / : / Glycoside hydrolase, family 24 / Phage lysozyme ...Transducin (heterotrimeric G protein), gamma chain / G Protein Gi Gamma 2 / Beta 2 adrenoceptor / Adrenoceptor family / G-protein alpha subunit, group S / Endolysin T4 type / T4-type lysozyme / : / Glycoside hydrolase, family 24 / Phage lysozyme / Lysozyme domain superfamily / YVTN repeat-like/Quinoprotein amine dehydrogenase / Serpentine type 7TM GPCR chemoreceptor Srsx / 7 Propeller / Methylamine Dehydrogenase; Chain H / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G-alpha domain profile. / G protein alpha subunit / Few Secondary Structures / Irregular / G-protein, gamma subunit / G-protein gamma subunit domain profile. / G-protein gamma-like domain / G-protein gamma-like domain superfamily / GGL domain / G protein gamma subunit-like motifs / GGL domain / Guanine nucleotide-binding protein, beta subunit / G-protein, beta subunit / G-protein coupled receptors family 1 signature. / Lysozyme-like domain superfamily / G protein-coupled receptor, rhodopsin-like / GPCR, rhodopsin-like, 7TM / G-protein coupled receptors family 1 profile. / 7 transmembrane receptor (rhodopsin family) / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / P-loop containing nucleotide triphosphate hydrolases / WD domain, G-beta repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / Rossmann fold / P-loop containing nucleoside triphosphate hydrolase / 3-Layer(aba) Sandwich / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
Chem-P0G / Endolysin / Beta-2 adrenergic receptor / Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
Similarity search - Component
Biological speciesHomo sapiens (human)
Enterobacteria phage RB59 (virus)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 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 1.3Oct 16, 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: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
B: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
G: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
R: Endolysin,Beta-2 adrenergic receptor chimera
N: Nanobody 35
hetero molecules


Theoretical massNumber of molelcules
Total (without water)163,8636
Polymers163,4935
Non-polymers3701
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, immunoprecipitation, microscopy, fluorescence resonance energy transfer
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area12970 Å2
ΔGint-66 kcal/mol
Surface area47050 Å2

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Components

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Guanine nucleotide-binding protein ... , 3 types, 3 molecules ABG

#1: Protein Guanine nucleotide-binding protein G(s) subunit alpha isoforms short / Adenylate cyclase-stimulating G alpha protein


Mass: 44326.160 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNAS, GNAS1, GSP / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P63092
#2: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Transducin beta chain 1


Mass: 38534.062 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNB1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P62873
#3: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / G gamma-I


Mass: 7861.143 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNG2 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P59768

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Protein / Antibody / Non-polymers , 3 types, 3 molecules RN

#4: Protein Endolysin,Beta-2 adrenergic receptor chimera / Lysis protein / Lysozyme / Muramidase / Beta-2 adrenoreceptor / Beta-2 adrenoceptor


Mass: 57630.762 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Enterobacteria phage RB59 (virus), (gene. exp.) Homo sapiens (human)
Gene: e, RB59_126, ADRB2, ADRB2R, B2AR / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: A0A097J809, UniProt: P07550, lysozyme
#5: Antibody Nanobody 35


Mass: 15140.742 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)
#6: Chemical ChemComp-P0G / 8-[(1R)-2-{[1,1-dimethyl-2-(2-methylphenyl)ethyl]amino}-1-hydroxyethyl]-5-hydroxy-2H-1,4-benzoxazin-3(4H)-one


Mass: 370.442 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C21H26N2O4 / Feature type: SUBJECT OF INVESTIGATION

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Details

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
1B2V2R-Gs protein subcomplex of a GPCR-G protein-beta-arrestin mega-complexCOMPLEX#1-#50MULTIPLE SOURCES
2B2V2R-Gs protein subcomplexCOMPLEX#1-#41RECOMBINANT
3Nanobody 35COMPLEX#51RECOMBINANT
Molecular weightUnits: MEGADALTONS / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Lama glama (llama)9844
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Trichoplusia ni (cabbage looper)7111
23Escherichia coli (E. coli)562
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

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: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 299893 / Symmetry type: POINT

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