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- PDB-9ee9: Cryo-EM structure of the adenosine A2A receptor intermediate boun... -

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Basic information

Entry
Database: PDB / ID: 9ee9
TitleCryo-EM structure of the adenosine A2A receptor intermediate bound to a miniGs heterotrimer
Components
  • (Guanine nucleotide-binding protein ...) x 3
  • Adenosine receptor A2a
  • nanobody Nb35
KeywordsSIGNALING PROTEIN/IMMUNE SYSTEM / GPCR / Heterotrimer / Receptor / cryo-EM / SIGNALING PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


regulation of norepinephrine secretion / positive regulation of acetylcholine secretion, neurotransmission / negative regulation of alpha-beta T cell activation / positive regulation of circadian sleep/wake cycle, sleep / Adenosine P1 receptors / G protein-coupled adenosine receptor activity / response to purine-containing compound / G protein-coupled adenosine receptor signaling pathway / NGF-independant TRKA activation / Surfactant metabolism ...regulation of norepinephrine secretion / positive regulation of acetylcholine secretion, neurotransmission / negative regulation of alpha-beta T cell activation / positive regulation of circadian sleep/wake cycle, sleep / Adenosine P1 receptors / G protein-coupled adenosine receptor activity / response to purine-containing compound / G protein-coupled adenosine receptor signaling pathway / NGF-independant TRKA activation / Surfactant metabolism / sensory perception / positive regulation of urine volume / synaptic transmission, dopaminergic / type 5 metabotropic glutamate receptor binding / negative regulation of vascular permeability / synaptic transmission, cholinergic / positive regulation of glutamate secretion / intermediate filament / presynaptic active zone / blood circulation / response to caffeine / eating behavior / inhibitory postsynaptic potential / alpha-actinin binding / regulation of calcium ion transport / asymmetric synapse / PKA activation in glucagon signalling / axolemma / membrane depolarization / developmental growth / hair follicle placode formation / phagocytosis / D1 dopamine receptor binding / cellular defense response / prepulse inhibition / positive regulation of synaptic transmission, glutamatergic / intracellular transport / vascular endothelial cell response to laminar fluid shear stress / renal water homeostasis / activation of adenylate cyclase activity / Hedgehog 'off' state / adenylate cyclase-activating adrenergic receptor signaling pathway / neuron projection morphogenesis / cellular response to glucagon stimulus / astrocyte activation / presynaptic modulation of chemical synaptic transmission / regulation of insulin secretion / response to amphetamine / adenylate cyclase activator activity / central nervous system development / trans-Golgi network membrane / positive regulation of long-term synaptic potentiation / positive regulation of apoptotic signaling pathway / positive regulation of synaptic transmission, GABAergic / regulation of mitochondrial membrane potential / positive regulation of protein secretion / excitatory postsynaptic potential / synaptic transmission, glutamatergic / locomotory behavior / apoptotic signaling pathway / negative regulation of inflammatory response to antigenic stimulus / bone development / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / platelet aggregation / negative regulation of inflammatory response / G-protein beta/gamma-subunit complex binding / vasodilation / 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 / 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 / blood coagulation / 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 / 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
Similarity search - Function
Adenosine A2A receptor / Adenosine receptor / 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 ...Adenosine A2A receptor / Adenosine receptor / 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
ADENOSINE / Adenosine receptor A2a / 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)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.16 Å
AuthorsBi, M. / Wang, X. / Ye, L. / Cheng, Y.
Funding support United States, 2items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM149659 United States
CitationJournal: Nat Commun / Year: 2025
Title: Structure and function of a near fully-activated intermediate GPCR-Gαβγ complex.
Authors: Maxine Bi / Xudong Wang / Jinan Wang / Jun Xu / Wenkai Sun / Victor Ayo Adediwura / Yinglong Miao / Yifan Cheng / Libin Ye /
Abstract: Unraveling the signaling roles of intermediate complexes is pivotal for G protein-coupled receptor (GPCR) drug development. Despite hundreds of GPCR-Gαβγ structures, these snapshots primarily ...Unraveling the signaling roles of intermediate complexes is pivotal for G protein-coupled receptor (GPCR) drug development. Despite hundreds of GPCR-Gαβγ structures, these snapshots primarily capture the fully activated complex. Consequently, the functions of intermediate GPCR-G protein complexes remain elusive. Guided by a conformational landscape visualized via F quantitative NMR and molecular dynamics (MD) simulations, we determined the structure of an intermediate GPCR-mini-Gαβγ complex at 2.6 Å using cryo-EM, by blocking its transition to the fully activated complex. Furthermore, we present direct evidence that the complex at this intermediate state initiates a rate-limited nucleotide exchange before transitioning to the fully activated complex. In this state, BODIPY-GDP/GTP based nucleotide exchange assays further indicated the α-helical domain of the Gα is partially open, allowing it to grasp a nucleotide at a non-canonical binding site, distinct from the canonical nucleotide-binding site. These advances bridge a significant gap in our understanding of the complexity of GPCR signaling.
History
DepositionNov 18, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 9, 2025Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Adenosine receptor A2a
B: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
C: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
D: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
E: nanobody Nb35
hetero molecules


Theoretical massNumber of molelcules
Total (without water)133,0766
Polymers132,8095
Non-polymers2671
Water00
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 BCD

#2: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Transducin beta chain 1


Mass: 37728.152 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNB1 / Production host: Spodoptera frugiperda (fall armyworm) / 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: Spodoptera frugiperda (fall armyworm) / References: UniProt: P59768
#4: Protein Guanine nucleotide-binding protein G(s) subunit alpha isoforms short / Adenylate cyclase-stimulating G alpha protein


Mass: 30776.713 Da / Num. of mol.: 1 / Fragment: UNP residues 5-64,204-394
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNAS, GNAS1, GSP / Production host: Escherichia coli (E. coli)
References: UniProt: P63092, Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement

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

#1: Protein Adenosine receptor A2a


Mass: 39517.078 Da / Num. of mol.: 1 / Mutation: V229C, R291A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ADORA2A, ADORA2 / Production host: Komagataella pastoris (fungus) / Strain (production host): SMD1163 / References: UniProt: P29274
#5: Antibody nanobody Nb35


Mass: 16926.076 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-ADN / ADENOSINE


Mass: 267.241 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H13N5O4 / 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: CELL / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Cryo-EM structure of the GPCR bound to a miniGs heterotrimer
Type: COMPLEX / Entity ID: #1-#5 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.144 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Komagataella pastoris (fungus)
Buffer solutionpH: 7.4
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 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 47.7 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.16 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 43472 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0027771
ELECTRON MICROSCOPYf_angle_d0.53610598
ELECTRON MICROSCOPYf_dihedral_angle_d3.9991097
ELECTRON MICROSCOPYf_chiral_restr0.041237
ELECTRON MICROSCOPYf_plane_restr0.0031353

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