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- PDB-7v69: Cryo-EM structure of a class A GPCR-G protein complex -

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Basic information

Entry
Database: PDB / ID: 7v69
TitleCryo-EM structure of a class A GPCR-G protein complex
Components
  • 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(i) subunit alpha-1
  • Muscarinic acetylcholine receptor M4
  • scFv16
KeywordsMEMBRANE PROTEIN / Complex
Function / homology
Function and homology information


Muscarinic acetylcholine receptors / G protein-coupled acetylcholine receptor activity / adenylate cyclase-inhibiting G protein-coupled acetylcholine receptor signaling pathway / G protein-coupled serotonin receptor activity / regulation of locomotion / G protein-coupled receptor signaling pathway, coupled to cyclic nucleotide second messenger / Adenylate cyclase inhibitory pathway / positive regulation of protein localization to cell cortex / regulation of cAMP-mediated signaling / D2 dopamine receptor binding ...Muscarinic acetylcholine receptors / G protein-coupled acetylcholine receptor activity / adenylate cyclase-inhibiting G protein-coupled acetylcholine receptor signaling pathway / G protein-coupled serotonin receptor activity / regulation of locomotion / G protein-coupled receptor signaling pathway, coupled to cyclic nucleotide second messenger / Adenylate cyclase inhibitory pathway / positive regulation of protein localization to cell cortex / regulation of cAMP-mediated signaling / D2 dopamine receptor binding / G protein-coupled serotonin receptor binding / regulation of mitotic spindle organization / cellular response to forskolin / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / Regulation of insulin secretion / G protein-coupled receptor binding / Olfactory Signaling Pathway / G-protein beta/gamma-subunit complex binding / Activation of the phototransduction cascade / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / G-protein activation / G protein-coupled acetylcholine receptor signaling pathway / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / Prostacyclin signalling through prostacyclin receptor / Glucagon signaling in metabolic regulation / G beta:gamma signalling through CDC42 / ADP signalling through P2Y purinoceptor 12 / G beta:gamma signalling through BTK / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / Sensory perception of sweet, bitter, and umami (glutamate) taste / photoreceptor disc membrane / response to peptide hormone / Adrenaline,noradrenaline inhibits insulin secretion / Glucagon-type ligand receptors / Vasopressin regulates renal water homeostasis via Aquaporins / G alpha (z) signalling events / cellular response to catecholamine stimulus / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / ADORA2B mediated anti-inflammatory cytokines production / adenylate cyclase-activating dopamine receptor signaling pathway / ADP signalling through P2Y purinoceptor 1 / G beta:gamma signalling through PI3Kgamma / cellular response to prostaglandin E stimulus / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / sensory perception of taste / GPER1 signaling / G-protein beta-subunit binding / GDP binding / Inactivation, recovery and regulation of the phototransduction cascade / heterotrimeric G-protein complex / G alpha (12/13) signalling events / extracellular vesicle / signaling receptor complex adaptor activity / Thrombin signalling through proteinase activated receptors (PARs) / retina development in camera-type eye / GTPase binding / Ca2+ pathway / phospholipase C-activating G protein-coupled receptor signaling pathway / cell cortex / midbody / G alpha (i) signalling events / fibroblast proliferation / G alpha (s) signalling events / chemical synaptic transmission / G alpha (q) signalling events / postsynaptic membrane / Ras protein signal transduction / cell population proliferation / Extra-nuclear estrogen signaling / cell surface receptor signaling pathway / cell cycle / G protein-coupled receptor signaling pathway / lysosomal membrane / cell division / GTPase activity / centrosome / dendrite / synapse / protein-containing complex binding / GTP binding / nucleolus / magnesium ion binding / signal transduction / extracellular exosome / nucleoplasm / membrane / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Muscarinic acetylcholine receptor M4 / Muscarinic acetylcholine receptor family / G-protein alpha subunit, group I / Serpentine type 7TM GPCR chemoreceptor Srsx / G-alpha domain profile. / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G protein alpha subunit / G-protein, gamma subunit ...Muscarinic acetylcholine receptor M4 / Muscarinic acetylcholine receptor family / G-protein alpha subunit, group I / Serpentine type 7TM GPCR chemoreceptor Srsx / G-alpha domain profile. / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G protein alpha subunit / G-protein, gamma subunit / G-protein gamma subunit domain profile. / GGL domain / G-protein gamma-like domain superfamily / G-protein gamma-like domain / GGL domain / G protein gamma subunit-like motifs / 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. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Muscarinic acetylcholine receptor M4 / 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(i) subunit alpha-1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsWang, J.J. / Wu, M. / Wu, L.J. / Hua, T. / Liu, Z.J. / Wang, T.
Funding support China, 2items
OrganizationGrant numberCountry
National Science Foundation (NSF, China)32022038 China
Chinese Academy of SciencesXDB37030104 China
CitationJournal: Nat Commun / Year: 2022
Title: The unconventional activation of the muscarinic acetylcholine receptor M4R by diverse ligands.
Authors: Jingjing Wang / Meng Wu / Zhangcheng Chen / Lijie Wu / Tian Wang / Dongmei Cao / Huan Wang / Shenhui Liu / Yueming Xu / Fei Li / Junlin Liu / Na Chen / Suwen Zhao / Jianjun Cheng / Sheng Wang / Tian Hua /
Abstract: Muscarinic acetylcholine receptors (mAChRs) respond to the neurotransmitter acetylcholine and play important roles in human nervous system. Muscarinic receptor 4 (M4R) is a promising drug target for ...Muscarinic acetylcholine receptors (mAChRs) respond to the neurotransmitter acetylcholine and play important roles in human nervous system. Muscarinic receptor 4 (M4R) is a promising drug target for treating neurological and mental disorders, such as Alzheimer's disease and schizophrenia. However, the lack of understanding on M4R's activation by subtype selective agonists hinders its therapeutic applications. Here, we report the structural characterization of M4R selective allosteric agonist, compound-110, as well as agonist iperoxo and positive allosteric modulator LY2119620. Our cryo-electron microscopy structures of compound-110, iperoxo or iperoxo-LY2119620 bound M4R-G complex reveal their different interaction modes and activation mechanisms of M4R, and the M4R-ip-LY-G structure validates the cooperativity between iperoxo and LY2119620 on M4R. Through the comparative structural and pharmacological analysis, compound-110 mostly occupies the allosteric binding pocket with vertical binding pose. Such a binding and activation mode facilitates its allostersic selectivity and agonist profile. In addition, in our schizophrenia-mimic mouse model study, compound-110 shows antipsychotic activity with low extrapyramidal side effects. Thus, this study provides structural insights to develop next-generation antipsychotic drugs selectively targeting on mAChRs subtypes.
History
DepositionAug 20, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0May 11, 2022Provider: repository / Type: Initial release
Revision 1.1Nov 23, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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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(i) subunit alpha-1
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
S: scFv16
R: Muscarinic acetylcholine receptor M4


Theoretical massNumber of molelcules
Total (without water)152,5465
Polymers152,5465
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area10230 Å2
ΔGint-62 kcal/mol
Surface area48240 Å2

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Components

#1: Protein Guanine nucleotide-binding protein G(i) subunit alpha-1 / Adenylate cyclase-inhibiting G alpha protein


Mass: 40559.160 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNAI1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P63096
#2: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Transducin beta chain 1


Mass: 37285.734 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
Details: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
Source: (gene. exp.) Homo sapiens (human) / Gene: GNG2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P59768
#4: Antibody scFv16


Mass: 27784.896 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Spodoptera frugiperda (fall armyworm)
#5: Protein Muscarinic acetylcholine receptor M4 /


Mass: 39055.043 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: residues 240-372 deleted to improve the protein yield for complex building
Source: (gene. exp.) Homo sapiens (human) / Gene: CHRM4 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P08173

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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
1M4R-iperoxo and Gi protein complexCOMPLEXall0RECOMBINANT
2Gi proteinGi alpha subunitCOMPLEX#1-#31RECOMBINANT
3scFv16COMPLEX#41RECOMBINANT
4M4R-iperoxoCOMPLEX#51RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
32Homo sapiens (human)9606
43Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Spodoptera frugiperda (fall armyworm)7108
32Spodoptera frugiperda (fall armyworm)7108
43Spodoptera frugiperda (fall armyworm)7108
Buffer solutionpH: 7.5
SpecimenConc.: 1.87 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: NICKEL/TITANIUM / Grid mesh size: 300 divisions/in. / Grid type: Homemade
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: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2000 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 58 e/Å2 / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameCategory
7RELIONmodel fitting
9PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4814856
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 270892 / Symmetry type: POINT
Atomic model buildingB value: 64.2 / Protocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 5DSG

5dsg

Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0028722
ELECTRON MICROSCOPYf_angle_d0.56911859
ELECTRON MICROSCOPYf_dihedral_angle_d5.1641205
ELECTRON MICROSCOPYf_chiral_restr0.0421385
ELECTRON MICROSCOPYf_plane_restr0.0041483

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