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- PDB-8unl: CryoEM structure of beta-2-adrenergic receptor in complex with GT... -

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Basic information

Entry
Database: PDB / ID: 8unl
TitleCryoEM structure of beta-2-adrenergic receptor in complex with GTP-bound Gs heterotrimer (Class A)
Components
  • (Guanine nucleotide-binding protein ...) x 3
  • Beta-2 adrenergic receptor
KeywordsSIGNALING PROTEIN / GPCR / Adrenergic / Receptor / G protein
Function / homology
Function and homology information


beta2-adrenergic receptor activity / positive regulation of mini excitatory postsynaptic potential / AMPA selective glutamate receptor signaling pathway / norepinephrine-epinephrine-mediated vasodilation involved in regulation of systemic arterial blood pressure / adenylate cyclase-inhibiting adrenergic receptor signaling pathway / positive regulation of autophagosome maturation / heat generation / norepinephrine binding / Adrenoceptors / negative regulation of smooth muscle contraction ...beta2-adrenergic receptor activity / positive regulation of mini excitatory postsynaptic potential / AMPA selective glutamate receptor signaling pathway / norepinephrine-epinephrine-mediated vasodilation involved in regulation of systemic arterial blood pressure / adenylate cyclase-inhibiting adrenergic receptor signaling pathway / positive regulation of autophagosome maturation / heat generation / norepinephrine binding / Adrenoceptors / negative regulation of smooth muscle contraction / positive regulation of cardiac muscle cell contraction / positive regulation of lipophagy / negative regulation of G protein-coupled receptor signaling pathway / negative regulation of multicellular organism growth / response to psychosocial stress / adrenergic receptor signaling pathway / diet induced thermogenesis / endosome to lysosome transport / adenylate cyclase-activating G protein-coupled bile acid receptor signaling pathway / adenylate cyclase-activating serotonin receptor signaling pathway / positive regulation of cAMP/PKA signal transduction / regulation of skeletal muscle contraction / adenylate cyclase binding / smooth muscle contraction / negative regulation of cardiac muscle cell apoptotic process / PKA activation in glucagon signalling / hair follicle placode formation / developmental growth / intracellular transport / potassium channel regulator activity / positive regulation of bone mineralization / bone resorption / D1 dopamine receptor binding / neuronal dense core vesicle / 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 / cellular response to acidic pH / adenylate cyclase-activating adrenergic receptor signaling pathway / cellular response to glucagon stimulus / positive regulation of cardiac muscle cell apoptotic process / intracellular glucose homeostasis / receptor-mediated endocytosis / brown fat cell differentiation / response to cold / adenylate cyclase activator activity / positive regulation of insulin secretion involved in cellular response to glucose stimulus / trans-Golgi network membrane / negative regulation of inflammatory response to antigenic stimulus / response to prostaglandin E / clathrin-coated endocytic vesicle membrane / bone development / platelet aggregation / cognition / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / G-protein beta/gamma-subunit complex binding / cellular response to amyloid-beta / Olfactory Signaling Pathway / Activation of the phototransduction cascade / G protein-coupled acetylcholine receptor signaling pathway / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / positive regulation of insulin secretion / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / G-protein activation / Glucagon signaling in metabolic regulation / G beta:gamma signalling through CDC42 / Prostacyclin signalling through prostacyclin receptor / G beta:gamma signalling through BTK / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / photoreceptor disc membrane / ADP signalling through P2Y purinoceptor 12 / sensory perception of smell / mitotic spindle / 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 / 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 / adenylate cyclase-activating dopamine receptor signaling pathway / Cargo recognition for clathrin-mediated endocytosis / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / GPER1 signaling / cellular response to prostaglandin E stimulus / G-protein beta-subunit binding / heterotrimeric G-protein complex / G alpha (12/13) signalling events / Inactivation, recovery and regulation of the phototransduction cascade / positive regulation of cold-induced thermogenesis / extracellular vesicle
Similarity search - Function
Beta 2 adrenoceptor / Adrenoceptor family / G-protein alpha subunit, group S / Serpentine type 7TM GPCR chemoreceptor Srsx / G protein alpha subunit, helical insertion / G protein alpha subunit / Guanine nucleotide binding protein (G-protein), alpha subunit / G-protein alpha subunit / G-alpha domain profile. / G-protein, gamma subunit ...Beta 2 adrenoceptor / Adrenoceptor family / G-protein alpha subunit, group S / Serpentine type 7TM GPCR chemoreceptor Srsx / G protein alpha subunit, helical insertion / G protein alpha subunit / Guanine nucleotide binding protein (G-protein), alpha subunit / G-protein alpha subunit / G-alpha domain profile. / 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. / 7 transmembrane receptor (rhodopsin family) / G protein-coupled receptor, rhodopsin-like / GPCR, rhodopsin-like, 7TM / G-protein coupled receptors family 1 profile. / 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 / GUANOSINE-5'-TRIPHOSPHATE / 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: 3.3 Å
AuthorsPapasergi-Scott, M.M. / Skiniotis, G.
Funding support United States, 1items
OrganizationGrant numberCountry
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
DepositionOct 19, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 6, 2024Provider: repository / Type: Initial release
Revision 1.1Mar 13, 2024Group: Database references / Category: citation_author
Revision 1.2Mar 27, 2024Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.3Jun 12, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.4Oct 23, 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)142,2616
Polymers141,5294
Non-polymers7322
Water181
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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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


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

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Protein , 1 types, 1 molecules R

#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

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Non-polymers , 3 types, 3 molecules

#5: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, energy-carrying molecule*YM
#6: 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
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN
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 with GTP
Type: COMPLEX
Details: Combined datasets of complex plunge frozen at 5 sec, 10 sec, or 17 sec after GTP addition to the sample.
Entity ID: #1-#4 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 7.5
Details: GTP was added just prior to freezing at 5 sec, 10 sec, or 17 sec before plunging.
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K
Details: GTP was added just prior to freezing at 5 sec, 10 sec, or 17 sec before plunging.

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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: 2000 nm / Nominal defocus min: 400 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K3 (6k x 4k)
Details: The cryoEM map is the result of combining multiple datasets: cryo-EM imaging of the beta-2AR-Gs + GTP (5 sec) complex was performed on a Titan Krios electron microscope equipped with a K3 ...Details: The cryoEM map is the result of combining multiple datasets: cryo-EM imaging of the beta-2AR-Gs + GTP (5 sec) complex was performed on a Titan Krios electron microscope equipped with a K3 Summit direct electron detector (Gatan). The microscope was operated at 300 kV accelerating voltage, with a nominal magnification of 105,000x in counting mode resulting in a magnified pixel size of 0.8677 Angstrom. A total exposure of 60.48 electrons/ Angstrom^2 over 63 frames with defocus ranging from -1.0 - -2.0 micrometers was used. Cryo-EM imaging of beta-2AR-Gs + GTP (10 sec) complex was performed on four separate grids over three collection sessions. The microscope was operated at 300 kV accelerating voltage, with a magnification at the camera of 58,679x in counting mode resulting in a magnified pixel size of 0.8521 Angstrom. For the first and second grids, movies were obtained at an exposure rate of 21.13 electrons/Angstrum^2/sec with defocus ranging from -0.4 - -2.0 micrometers. The total exposure time was 2.717 sec over 77 frames per movie stack. For an additional collection of the first grid, movies were obtained at an exposure rate of 20.95 electrons/ Angstrum^2/sec with defocus ranging from -0.4 -2.0 micrometers. The total exposure time was 2.717 sec over 77 frames per movie stack. For the third and fourth grids, movies were obtained at an exposure rate of 30.71 electrons/ Angstrum^2/sec with defocus ranging from -0.5 - -1.6 micrometers. The total exposure time was 2.008 sec over 79 frames per movie stack. Cryo-EM imaging of beta-2AR-Gs + GTP (17 sec) was performed on a Titan Krios equipped with a post-column energy filter, with a magnification of 105,000x in counting mode resulting in a magnified pixel size of 0.8677 Angstrom. Movies were obtained at an exposure rate of 32.46 electrons/Angstrum^2/sec with defocus ranging from -0.4 - -0.9 micrometers. The total exposure time was 1.999 sec over 79 frames per movie stack.

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Processing

EM software
IDNameCategoryDetails
2SerialEMimage acquisition
9PHENIXmodel refinement
10Cootmodel refinement
12cryoSPARCfinal Euler assignment
13cryoSPARCclassification
14cryoSPARC3D reconstructionLocal refinement masking out micelle
CTF correctionType: NONE
Particle selectionNum. of particles selected: 19918625
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 45634
Details: Contribution of each dataset to this refined class: 58.8% 5 sec dataset; 26.3% 10 sec dataset; 15.0% 17 sec dataset
Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0026707
ELECTRON MICROSCOPYf_angle_d0.4249180
ELECTRON MICROSCOPYf_dihedral_angle_d8.382965
ELECTRON MICROSCOPYf_chiral_restr0.0391102
ELECTRON MICROSCOPYf_plane_restr0.0031145

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