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- PDB-7t8x: Cryo-EM structure of ACh-bound M2R-Go signaling complex in S1 state -

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Basic information

Entry
Database: PDB / ID: 7t8x
TitleCryo-EM structure of ACh-bound M2R-Go signaling complex in S1 state
Components
  • (Guanine nucleotide-binding protein ...) x 3
  • Antibody fragment
  • Muscarinic acetylcholine receptor M2,muscarinic acetylcholine receptor M2 chimera
KeywordsMEMBRANE PROTEIN / GPCR / signaling complex / muscarinic receptor / acetylcholine
Function / homology
Function and homology information


Muscarinic acetylcholine receptors / G protein-coupled acetylcholine receptor activity / phospholipase C-activating G protein-coupled acetylcholine receptor signaling pathway / cholinergic synapse / adenylate cyclase-inhibiting G protein-coupled acetylcholine receptor signaling pathway / regulation of smooth muscle contraction / mu-type opioid receptor binding / corticotropin-releasing hormone receptor 1 binding / arrestin family protein binding / vesicle docking involved in exocytosis ...Muscarinic acetylcholine receptors / G protein-coupled acetylcholine receptor activity / phospholipase C-activating G protein-coupled acetylcholine receptor signaling pathway / cholinergic synapse / adenylate cyclase-inhibiting G protein-coupled acetylcholine receptor signaling pathway / regulation of smooth muscle contraction / mu-type opioid receptor binding / corticotropin-releasing hormone receptor 1 binding / arrestin family protein binding / vesicle docking involved in exocytosis / G protein-coupled dopamine receptor signaling pathway / regulation of heart contraction / parallel fiber to Purkinje cell synapse / G protein-coupled receptor signaling pathway, coupled to cyclic nucleotide second messenger / postsynaptic modulation of chemical synaptic transmission / adenylate cyclase regulator activity / G protein-coupled serotonin receptor binding / adenylate cyclase-inhibiting serotonin receptor signaling pathway / presynaptic modulation of chemical synaptic transmission / muscle contraction / locomotory behavior / clathrin-coated endocytic vesicle membrane / negative regulation of insulin secretion / GABA-ergic synapse / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / G-protein beta/gamma-subunit complex binding / response to virus / 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 / 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 / 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 / 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 / nervous system development / heterotrimeric G-protein complex / G alpha (12/13) signalling events / sensory perception of taste / extracellular vesicle / presynapse / signaling receptor complex adaptor activity / Thrombin signalling through proteinase activated receptors (PARs) / Clathrin-mediated endocytosis / retina development in camera-type eye / G protein activity / presynaptic membrane / cell body / GTPase binding / Ca2+ pathway / fibroblast proliferation / High laminar flow shear stress activates signaling by PIEZO1 and PECAM1:CDH5:KDR in endothelial cells / G alpha (i) signalling events / G alpha (s) signalling events / phospholipase C-activating G protein-coupled receptor signaling pathway / G alpha (q) signalling events / chemical synaptic transmission / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / Ras protein signal transduction / postsynaptic membrane / Extra-nuclear estrogen signaling / cell population proliferation / cilium / ciliary basal body / G protein-coupled receptor signaling pathway / lysosomal membrane / GTPase activity / dendrite / synapse / GTP binding / protein-containing complex binding / nucleolus / glutamatergic synapse / Golgi apparatus / signal transduction
Similarity search - Function
Muscarinic acetylcholine receptor M2 / Muscarinic acetylcholine receptor family / G-protein alpha subunit, group I / 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 ...Muscarinic acetylcholine receptor M2 / Muscarinic acetylcholine receptor family / G-protein alpha subunit, group I / 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 / 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 / Immunoglobulins / Immunoglobulin-like / Sandwich / P-loop containing nucleoside triphosphate hydrolase / Mainly Beta
Similarity search - Domain/homology
ACETYLCHOLINE / Muscarinic acetylcholine receptor M2 / Guanine nucleotide-binding protein G(o) subunit alpha / 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
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.21 Å
AuthorsXu, J. / Wang, Q. / Du, Y. / Kobilka, B.K.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Commun / Year: 2023
Title: Structural and dynamic insights into supra-physiological activation and allosteric modulation of a muscarinic acetylcholine receptor.
Authors: Jun Xu / Qinggong Wang / Harald Hübner / Yunfei Hu / Xiaogang Niu / Haoqing Wang / Shoji Maeda / Asuka Inoue / Yuyong Tao / Peter Gmeiner / Yang Du / Changwen Jin / Brian K Kobilka /
Abstract: The M2 muscarinic receptor (M2R) is a prototypical G-protein-coupled receptor (GPCR) that serves as a model system for understanding GPCR regulation by both orthosteric and allosteric ligands. Here, ...The M2 muscarinic receptor (M2R) is a prototypical G-protein-coupled receptor (GPCR) that serves as a model system for understanding GPCR regulation by both orthosteric and allosteric ligands. Here, we investigate the mechanisms governing M2R signaling versatility using cryo-electron microscopy (cryo-EM) and NMR spectroscopy, focusing on the physiological agonist acetylcholine and a supra-physiological agonist iperoxo, as well as a positive allosteric modulator LY2119620. These studies reveal that acetylcholine stabilizes a more heterogeneous M2R-G-protein complex than iperoxo, where two conformers with distinctive G-protein orientations were determined. We find that LY2119620 increases the affinity for both agonists, but differentially modulates agonists efficacy in G-protein and β-arrestin pathways. Structural and spectroscopic analysis suggest that LY211620 stabilizes distinct intracellular conformational ensembles from agonist-bound M2R, which may enhance β-arrestin recruitment while impairing G-protein activation. These results highlight the role of conformational dynamics in the complex signaling behavior of GPCRs, and could facilitate design of better drugs.
History
DepositionDec 17, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 25, 2023Provider: repository / Type: Initial release
Revision 1.1Feb 7, 2024Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / 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
Revision 1.2Nov 6, 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

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Assembly

Deposited unit
A: Muscarinic acetylcholine receptor M2,muscarinic acetylcholine receptor M2 chimera
B: Guanine nucleotide-binding protein G(o) subunit alpha
C: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
D: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
E: Antibody fragment
hetero molecules


Theoretical massNumber of molelcules
Total (without water)152,9106
Polymers152,7645
Non-polymers1461
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
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(o) subunit alpha


Mass: 40104.551 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNAO1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P09471
#3: 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: Trichoplusia ni (cabbage looper) / References: UniProt: P62873
#4: 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 AE

#1: Protein Muscarinic acetylcholine receptor M2,muscarinic acetylcholine receptor M2 chimera


Mass: 39729.910 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CHRM2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P08172
#5: Antibody Antibody fragment


Mass: 27340.482 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Spodoptera frugiperda (fall armyworm)
#6: Chemical ChemComp-ACH / ACETYLCHOLINE


Mass: 146.207 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C7H16NO2 / 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

ComponentName: ACh-bound M2R-GoA-scFv16 complex / Type: COMPLEX / Entity ID: #1-#5 / Source: MULTIPLE SOURCES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
31Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Spodoptera frugiperda (fall armyworm)7108
31Trichoplusia ni (cabbage looper)7111
Buffer solutionpH: 7.5
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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 / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: NONE
3D reconstructionResolution: 3.21 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 127302 / Symmetry type: POINT

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