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- PDB-8fx5: Human M4 muscarinic acetylcholine receptor complex with Gi1 and x... -

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Basic information

Entry
Database: PDB / ID: 8fx5
TitleHuman M4 muscarinic acetylcholine receptor complex with Gi1 and xanomeline
Components
  • (Guanine nucleotide-binding protein ...) x 3
  • Antibody fragment scFv16
  • Muscarinic acetylcholine receptor M4
KeywordsMEMBRANE PROTEIN/SIGNALING PROTEIN / 7 transmembrane receptor / SIGNALING PROTEIN-IMMUNE SYSTEM complex / MEMBRANE PROTEIN / MEMBRANE PROTEIN-SIGNALING PROTEIN-IMMUNE SYSTEM complex / MEMBRANE PROTEIN-SIGNALING PROTEIN complex
Function / homology
Function and homology information


Adenylate cyclase inhibitory pathway / Adrenaline,noradrenaline inhibits insulin secretion / ADP signalling through P2Y purinoceptor 12 / Muscarinic acetylcholine receptors / Extra-nuclear estrogen signaling / G protein-coupled acetylcholine receptor activity / G alpha (i) signalling events / adenylate cyclase-inhibiting G protein-coupled acetylcholine receptor signaling pathway / G protein-coupled serotonin receptor activity / GTPase activating protein binding ...Adenylate cyclase inhibitory pathway / Adrenaline,noradrenaline inhibits insulin secretion / ADP signalling through P2Y purinoceptor 12 / Muscarinic acetylcholine receptors / Extra-nuclear estrogen signaling / G protein-coupled acetylcholine receptor activity / G alpha (i) signalling events / adenylate cyclase-inhibiting G protein-coupled acetylcholine receptor signaling pathway / G protein-coupled serotonin receptor activity / GTPase activating protein binding / negative regulation of synaptic transmission / regulation of locomotion / G protein-coupled receptor signaling pathway, coupled to cyclic nucleotide second messenger / 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 / G protein-coupled receptor binding / G-protein beta/gamma-subunit complex binding / Olfactory Signaling Pathway / Activation of the phototransduction cascade / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / 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 / sensory perception of taste / 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 / GPER1 signaling / GDP binding / 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) / GTPase binding / retina development in camera-type eye / phospholipase C-activating G protein-coupled receptor signaling pathway / Ca2+ pathway / cell cortex / midbody / G alpha (i) signalling events / fibroblast proliferation / G alpha (s) signalling events / G alpha (q) signalling events / chemical synaptic transmission / postsynaptic membrane / cell population proliferation / Ras protein signal transduction / Extra-nuclear estrogen signaling / cell surface receptor signaling pathway / cell cycle / G protein-coupled receptor signaling pathway / cell division / lysosomal membrane / GTPase activity / centrosome / synapse / dendrite / protein-containing complex binding / nucleolus / GTP binding / magnesium ion binding / signal transduction / protein-containing complex / extracellular exosome / nucleoplasm / membrane / plasma membrane / cytoplasm / cytosol
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. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD domain, G-beta repeat / 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
xanomeline / Guanine nucleotide-binding protein G(i) subunit alpha-1 / 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
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.45 Å
AuthorsVuckovic, Z. / Mobbs, J.I. / Glukhova, A. / Sexton, P.M. / Danev, R. / Thal, D.M.
Funding support United Kingdom, Australia, Japan, 7items
OrganizationGrant numberCountry
Wellcome Trust201529/Z/16/Z United Kingdom
National Health and Medical Research Council (NHMRC, Australia)1055134 Australia
National Health and Medical Research Council (NHMRC, Australia)1150083 Australia
National Health and Medical Research Council (NHMRC, Australia)1138448 Australia
Australian Research Council (ARC)DE170100152 Australia
Australian Research Council (ARC)IC200100052 Australia
Japan Science and Technology18069571 Japan
CitationJournal: Nat Commun / Year: 2023
Title: Xanomeline displays concomitant orthosteric and allosteric binding modes at the M mAChR.
Authors: Wessel A C Burger / Vi Pham / Ziva Vuckovic / Alexander S Powers / Jesse I Mobbs / Yianni Laloudakis / Alisa Glukhova / Denise Wootten / Andrew B Tobin / Patrick M Sexton / Steven M Paul / ...Authors: Wessel A C Burger / Vi Pham / Ziva Vuckovic / Alexander S Powers / Jesse I Mobbs / Yianni Laloudakis / Alisa Glukhova / Denise Wootten / Andrew B Tobin / Patrick M Sexton / Steven M Paul / Christian C Felder / Radostin Danev / Ron O Dror / Arthur Christopoulos / Celine Valant / David M Thal /
Abstract: The M muscarinic acetylcholine receptor (M mAChR) has emerged as a drug target of high therapeutic interest due to its expression in regions of the brain involved in the regulation of psychosis, ...The M muscarinic acetylcholine receptor (M mAChR) has emerged as a drug target of high therapeutic interest due to its expression in regions of the brain involved in the regulation of psychosis, cognition, and addiction. The mAChR agonist, xanomeline, has provided significant improvement in the Positive and Negative Symptom Scale (PANSS) scores in a Phase II clinical trial for the treatment of patients suffering from schizophrenia. Here we report the active state cryo-EM structure of xanomeline bound to the human M mAChR in complex with the heterotrimeric G transducer protein. Unexpectedly, two molecules of xanomeline were found to concomitantly bind to the monomeric M mAChR, with one molecule bound in the orthosteric (acetylcholine-binding) site and a second molecule in an extracellular vestibular allosteric site. Molecular dynamic simulations supports the structural findings, and pharmacological validation confirmed that xanomeline acts as a dual orthosteric and allosteric ligand at the human M mAChR. These findings provide a basis for further understanding xanomeline's complex pharmacology and highlight the myriad of ways through which clinically relevant ligands can bind to and regulate GPCRs.
History
DepositionJan 23, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 13, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
R: Muscarinic acetylcholine receptor M4
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
H: Antibody fragment scFv16
A: Guanine nucleotide-binding protein G(i) subunit alpha-1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)153,1747
Polymers152,6125
Non-polymers5632
Water84747
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 BGA

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


Mass: 38402.867 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: 7729.947 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
#5: Protein Guanine nucleotide-binding protein G(i) subunit alpha-1 / Adenylate cyclase-inhibiting G alpha protein


Mass: 40414.047 Da / Num. of mol.: 1 / Mutation: S47N, G203A, E245A, A326S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Gnai1, Gnai-1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: B2RSH2

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Protein / Antibody , 2 types, 2 molecules RH

#1: Protein Muscarinic acetylcholine receptor M4


Mass: 39598.277 Da / Num. of mol.: 1
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
#4: Antibody Antibody fragment scFv16


Mass: 26466.486 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Trichoplusia ni (cabbage looper)

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Non-polymers , 2 types, 49 molecules

#6: Chemical ChemComp-XNO / xanomeline / (5M)-5-[4-(hexyloxy)-1,2,5-thiadiazol-3-yl]-1-methyl-1,2,3,6-tetrahydropyridine


Mass: 281.417 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C14H23N3OS / Feature type: SUBJECT OF INVESTIGATION
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 47 / Source method: isolated from a natural source / Formula: H2O

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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

ComponentName: M4 mAChR bound to agonist xanomeline in complex with dominant negative Galpha-i1, Gbeta1, Ggamma2, and scFv16
Type: COMPLEX / Entity ID: #1-#5 / Source: MULTIPLE SOURCES
Buffer solutionpH: 7.5
SpecimenEmbedding 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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1500 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 52.4 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.45 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 395419 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0048957
ELECTRON MICROSCOPYf_angle_d0.82312150
ELECTRON MICROSCOPYf_dihedral_angle_d11.3593186
ELECTRON MICROSCOPYf_chiral_restr0.0551391
ELECTRON MICROSCOPYf_plane_restr0.0071540

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