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- PDB-8e9x: CryoEM structure of miniGo-coupled hM4Di in complex with DCZ -

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Basic information

Entry
Database: PDB / ID: 8e9x
TitleCryoEM structure of miniGo-coupled hM4Di in complex with DCZ
Components
  • (Guanine nucleotide-binding protein ...) x 2
  • Muscarinic acetylcholine receptor M4
  • miniGoYoplait
  • scFv16
KeywordsMEMBRANE PROTEIN / GPCR / DCZ / active state / hM4Di / DREADD
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 / mu-type opioid receptor binding / corticotropin-releasing hormone receptor 1 binding / G protein-coupled serotonin receptor activity / regulation of locomotion / dopamine receptor signaling pathway / G protein-coupled receptor signaling pathway, coupled to cyclic nucleotide second messenger / G protein-coupled serotonin receptor binding ...Muscarinic acetylcholine receptors / G protein-coupled acetylcholine receptor activity / adenylate cyclase-inhibiting G protein-coupled acetylcholine receptor signaling pathway / mu-type opioid receptor binding / corticotropin-releasing hormone receptor 1 binding / G protein-coupled serotonin receptor activity / regulation of locomotion / dopamine receptor signaling pathway / G protein-coupled receptor signaling pathway, coupled to cyclic nucleotide second messenger / G protein-coupled serotonin receptor binding / muscle contraction / 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 / 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 / ADP signalling through P2Y purinoceptor 1 / adenylate cyclase-activating dopamine receptor signaling pathway / 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 / 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 / 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 / G protein-coupled receptor signaling pathway / lysosomal membrane / GTPase activity / synapse / dendrite / protein-containing complex binding / GTP binding / signal transduction / extracellular exosome / membrane / metal ion binding / 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
Chem-WEC / cDNA FLJ31446 fis, clone NT2NE2000909, highly similar to Guanine nucleotide-binding protein G(o) subunit alpha 1 / Muscarinic acetylcholine receptor M4 / 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: 2.7 Å
AuthorsZhang, S. / Fay, J.F. / Roth, B.L.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)U24DK1169195 United States
National Institutes of Health/National Institute of Mental Health (NIH/NIMH)Psychoactive Drug Screening (PDSP) United States
CitationJournal: Nature / Year: 2022
Title: Molecular basis for selective activation of DREADD-based chemogenetics.
Authors: Shicheng Zhang / Ryan H Gumpper / Xi-Ping Huang / Yongfeng Liu / Brian E Krumm / Can Cao / Jonathan F Fay / Bryan L Roth /
Abstract: Designer receptors exclusively activated by designer drugs (DREADDs) represent a powerful chemogenetic technology for the remote control of neuronal activity and cellular signalling. The muscarinic ...Designer receptors exclusively activated by designer drugs (DREADDs) represent a powerful chemogenetic technology for the remote control of neuronal activity and cellular signalling. The muscarinic receptor-based DREADDs are the most widely used chemogenetic tools in neuroscience research. The G-coupled DREADD (hM3Dq) is used to enhance neuronal activity, whereas the G-coupled DREADD (hM4Di) is utilized to inhibit neuronal activity. Here we report four DREADD-related cryogenic electron microscopy high-resolution structures: a hM3Dq-miniG complex and a hM4Di-miniG complex bound to deschloroclozapine; a hM3Dq-miniG complex bound to clozapine-N-oxide; and a hM3R-miniG complex bound to iperoxo. Complemented with mutagenesis, functional and computational simulation data, our structures reveal key details of the recognition of DREADD chemogenetic actuators and the molecular basis for activation. These findings should accelerate the structure-guided discovery of next-generation chemogenetic tools.
History
DepositionAug 27, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 30, 2022Provider: repository / Type: Initial release
Revision 1.1Dec 14, 2022Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Dec 21, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Muscarinic acetylcholine receptor M4
B: miniGo
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: scFv16
hetero molecules


Theoretical massNumber of molelcules
Total (without water)136,5046
Polymers136,2125
Non-polymers2921
Water0
1


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

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Components

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Protein , 2 types, 2 molecules AB

#1: Protein Muscarinic acetylcholine receptor M4 /


Mass: 39225.297 Da / Num. of mol.: 1 / Mutation: Y113C, A203G
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CHRM4 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P08173
#2: Protein miniGo / Yoplait


Mass: 25159.777 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNAO1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: B3KP89, UniProt: P09471

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Guanine nucleotide-binding protein ... , 2 types, 2 molecules CD

#3: 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
#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: Spodoptera frugiperda (fall armyworm) / References: UniProt: P59768

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Antibody / Non-polymers , 2 types, 2 molecules E

#5: Antibody scFv16


Mass: 26679.721 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-WEC / 11-(4-methylpiperazin-1-yl)-5H-dibenzo[b,e][1,4]diazepine


Mass: 292.378 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C18H20N4 / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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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
1Gq-coupled hM3R complexCOMPLEX#1-#50RECOMBINANT
2muscarinic acetylcholine receptor 4, miniGo protein, Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1, Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2COMPLEX#1-#41RECOMBINANT
3Single-chain variable fragment scFv16COMPLEX#51RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Spodoptera frugiperda (fall armyworm)7108
23Spodoptera frugiperda (fall armyworm)7108
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2900 nm / Nominal defocus min: 200 nm
Image recordingElectron dose: 46.1 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 2982

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Processing

SoftwareName: PHENIX / Version: 1.19_4092: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 435476 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0028565
ELECTRON MICROSCOPYf_angle_d0.46811670
ELECTRON MICROSCOPYf_dihedral_angle_d3.9921190
ELECTRON MICROSCOPYf_chiral_restr0.041362
ELECTRON MICROSCOPYf_plane_restr0.0031470

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