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- PDB-8geb: CryoEM structure of beta-2-adrenergic receptor in complex with nu... -

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Basic information

Entry
Database: PDB / ID: 8geb
TitleCryoEM structure of beta-2-adrenergic receptor in complex with nucleotide-free Gs heterotrimer (#12 of 20)
Components
  • 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
KeywordsSIGNALING PROTEIN / GPCR / Adrenergic / Receptor / G protein
Function / homology
Function and homology information


positive regulation of mini excitatory postsynaptic potential / beta2-adrenergic receptor activity / negative regulation of smooth muscle contraction / AMPA selective glutamate receptor signaling pathway / positive regulation of autophagosome maturation / norepinephrine-epinephrine-mediated vasodilation involved in regulation of systemic arterial blood pressure / heat generation / norepinephrine binding / Adrenoceptors / positive regulation of lipophagy ...positive regulation of mini excitatory postsynaptic potential / beta2-adrenergic receptor activity / negative regulation of smooth muscle contraction / AMPA selective glutamate receptor signaling pathway / positive regulation of autophagosome maturation / norepinephrine-epinephrine-mediated vasodilation involved in regulation of systemic arterial blood pressure / heat generation / norepinephrine binding / Adrenoceptors / positive regulation of lipophagy / negative regulation of multicellular organism growth / negative regulation of G protein-coupled receptor signaling pathway / endosome to lysosome transport / adrenergic receptor signaling pathway / response to psychosocial stress / diet induced thermogenesis / positive regulation of cAMP/PKA signal transduction / adenylate cyclase binding / smooth muscle contraction / PKA activation in glucagon signalling / developmental growth / hair follicle placode formation / bone resorption / D1 dopamine receptor binding / potassium channel regulator activity / positive regulation of bone mineralization / neuronal dense core vesicle / intracellular transport / brown fat cell differentiation / vascular endothelial cell response to laminar fluid shear stress / renal water homeostasis / activation of adenylate cyclase activity / Hedgehog 'off' state / intercellular bridge / regulation of sodium ion transport / adenylate cyclase-activating adrenergic receptor signaling pathway / cellular response to glucagon stimulus / regulation of insulin secretion / receptor-mediated endocytosis / response to cold / adenylate cyclase activator activity / 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 / cognition / Olfactory Signaling Pathway / 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 / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / G-protein activation / cellular response to amyloid-beta / Prostacyclin signalling through prostacyclin receptor / G beta:gamma signalling through CDC42 / Glucagon signaling in metabolic regulation / G beta:gamma signalling through BTK / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / ADP signalling through P2Y purinoceptor 12 / photoreceptor disc membrane / Sensory perception of sweet, bitter, and umami (glutamate) taste / Glucagon-type ligand receptors / Adrenaline,noradrenaline inhibits insulin secretion / Vasopressin regulates renal water homeostasis via Aquaporins / mitotic spindle / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / G alpha (z) signalling events / cellular response to catecholamine stimulus / ADP signalling through P2Y purinoceptor 1 / ADORA2B mediated anti-inflammatory cytokines production / G beta:gamma signalling through PI3Kgamma / sensory perception of smell / adenylate cyclase-activating dopamine receptor signaling pathway / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / GPER1 signaling / Inactivation, recovery and regulation of the phototransduction cascade / Cargo recognition for clathrin-mediated endocytosis / cellular response to prostaglandin E stimulus / G-protein beta-subunit binding / heterotrimeric G-protein complex / G alpha (12/13) signalling events / sensory perception of taste / extracellular vesicle / signaling receptor complex adaptor activity / Thrombin signalling through proteinase activated receptors (PARs) / Clathrin-mediated endocytosis / positive regulation of cold-induced thermogenesis / amyloid-beta binding / retina development in camera-type eye / G protein activity / microtubule cytoskeleton / GTPase binding / Ca2+ pathway
Similarity search - Function
Beta 2 adrenoceptor / Adrenoceptor family / G-protein alpha subunit, group S / Serpentine type 7TM GPCR chemoreceptor Srsx / 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 / G-protein, gamma subunit ...Beta 2 adrenoceptor / Adrenoceptor family / G-protein alpha subunit, group S / Serpentine type 7TM GPCR chemoreceptor Srsx / 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 / 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 / G protein beta WD-40 repeat protein / Guanine nucleotide-binding protein, beta subunit / G-protein, beta subunit / 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) / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / 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 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Chem-G1I / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsPapasergi-Scott, M.M. / Skiniotis, G.
Funding support United States, 2items
OrganizationGrant numberCountry
Other government
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS) United States
CitationJournal: Nature / Year: 2024
Title: Time-resolved cryo-EM of G-protein activation by a GPCR.
Authors: Makaía M Papasergi-Scott / Guillermo Pérez-Hernández / Hossein Batebi / Yang Gao / Gözde Eskici / Alpay B Seven / Ouliana Panova / Daniel Hilger / Marina Casiraghi / Feng He / Luis Maul ...Authors: Makaía M Papasergi-Scott / Guillermo Pérez-Hernández / Hossein Batebi / Yang Gao / Gözde Eskici / Alpay B Seven / Ouliana Panova / Daniel Hilger / Marina Casiraghi / Feng He / Luis Maul / Peter Gmeiner / Brian K Kobilka / Peter W Hildebrand / Georgios Skiniotis /
Abstract: G-protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by stimulating guanine nucleotide exchange in the Gα subunit. To visualize this mechanism, we developed a time-resolved cryo-EM ...G-protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by stimulating guanine nucleotide exchange in the Gα subunit. To visualize this mechanism, we developed a time-resolved cryo-EM approach that examines the progression of ensembles of pre-steady-state intermediates of a GPCR-G-protein complex. By monitoring the transitions of the stimulatory G protein in complex with the β-adrenergic receptor at short sequential time points after GTP addition, we identified the conformational trajectory underlying G-protein activation and functional dissociation from the receptor. Twenty structures generated from sequential overlapping particle subsets along this trajectory, compared to control structures, provide a high-resolution description of the order of main events driving G-protein activation in response to GTP binding. Structural changes propagate from the nucleotide-binding pocket and extend through the GTPase domain, enacting alterations to Gα switch regions and the α5 helix that weaken the G-protein-receptor interface. Molecular dynamics simulations with late structures in the cryo-EM trajectory support that enhanced ordering of GTP on closure of the α-helical domain against the nucleotide-bound Ras-homology domain correlates with α5 helix destabilization and eventual dissociation of the G protein from the GPCR. These findings also highlight the potential of time-resolved cryo-EM as a tool for mechanistic dissection of GPCR signalling events.
History
DepositionMar 6, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 6, 2024Provider: repository / Type: Initial release
Revision 1.1Mar 27, 2024Group: Database references / Category: citation / citation_author / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jun 5, 2024Group: 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 13, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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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: Beta-2 adrenergic receptor
hetero molecules


Theoretical massNumber of molelcules
Total (without water)141,7385
Polymers141,5294
Non-polymers2091
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#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


Mass: 37573.988 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
#4: Protein Beta-2 adrenergic receptor / Beta-2 adrenoreceptor / Beta-2 adrenoceptor


Mass: 51767.242 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ADRB2, ADRB2R, B2AR / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P07550
#5: Chemical ChemComp-G1I / (5R,6R)-6-(methylamino)-5,6,7,8-tetrahydronaphthalene-1,2,5-triol


Mass: 209.242 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C11H15NO3 / Feature type: SUBJECT OF INVESTIGATION
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

ComponentName: Complex of beta-2 adrenergic receptor and Gs heterotrimer
Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 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 defocus max: 2500 nm / Nominal defocus min: 1200 nm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 52 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameCategory
2SerialEMimage acquisition
9PHENIXmodel refinement
10Cootmodel refinement
12cryoSPARCfinal Euler assignment
13cryoSPARCclassification
14cryoSPARC3D reconstruction
CTF correctionType: NONE
Particle selectionNum. of particles selected: 4190258
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 105147 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0036375
ELECTRON MICROSCOPYf_angle_d0.4888736
ELECTRON MICROSCOPYf_dihedral_angle_d3.959947
ELECTRON MICROSCOPYf_chiral_restr0.0391052
ELECTRON MICROSCOPYf_plane_restr0.0031108

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