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- PDB-8k9l: Full agonist- and positive allosteric modulator-bound mu-type opi... -

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Basic information

Entry
Database: PDB / ID: 8k9l
TitleFull agonist- and positive allosteric modulator-bound mu-type opioid receptor-G protein complex
Components
  • (Guanine nucleotide-binding protein ...) x 2
  • DAMGO
  • G protein subunit alpha i3
  • Soluble cytochrome b562,Mu-type opioid receptor,Mu-type opioid receptor
  • scFv16
KeywordsSIGNALING PROTEIN
Function / homology
Function and homology information


Opioid Signalling / beta-endorphin receptor activity / morphine receptor activity / negative regulation of Wnt protein secretion / regulation of cellular response to stress / G protein-coupled opioid receptor signaling pathway / behavioral response to ethanol / positive regulation of cAMP-mediated signaling / adenylate cyclase-inhibiting G protein-coupled acetylcholine receptor signaling pathway / sensory perception ...Opioid Signalling / beta-endorphin receptor activity / morphine receptor activity / negative regulation of Wnt protein secretion / regulation of cellular response to stress / G protein-coupled opioid receptor signaling pathway / behavioral response to ethanol / positive regulation of cAMP-mediated signaling / adenylate cyclase-inhibiting G protein-coupled acetylcholine receptor signaling pathway / sensory perception / negative regulation of nitric oxide biosynthetic process / neuropeptide binding / negative regulation of cAMP-mediated signaling / GTP metabolic process / regulation of NMDA receptor activity / positive regulation of neurogenesis / G protein-coupled dopamine receptor signaling pathway / negative regulation of cytosolic calcium ion concentration / G protein-coupled receptor signaling pathway, coupled to cyclic nucleotide second messenger / positive regulation of macroautophagy / G-protein alpha-subunit binding / voltage-gated calcium channel activity / neuropeptide signaling pathway / MECP2 regulates neuronal receptors and channels / sensory perception of pain / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / Peptide ligand-binding receptors / G protein-coupled receptor binding / G protein-coupled receptor activity / 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 / positive regulation of nitric oxide biosynthetic process / 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 / midbody / G alpha (i) signalling events / positive regulation of cytosolic calcium ion concentration / fibroblast proliferation / G alpha (s) signalling events / G alpha (q) signalling events / perikaryon / Interleukin-4 and Interleukin-13 signaling / cell population proliferation / Ras protein signal transduction / Extra-nuclear estrogen signaling / positive regulation of ERK1 and ERK2 cascade / endosome / neuron projection / G protein-coupled receptor signaling pathway / negative regulation of cell population proliferation / cell division / lysosomal membrane / axon / Golgi membrane / GTPase activity / centrosome / synapse / dendrite / protein-containing complex binding
Similarity search - Function
Mu opioid receptor / Opioid receptor / G-protein alpha subunit, group I / 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. ...Mu opioid receptor / Opioid receptor / G-protein alpha subunit, group I / 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
DAMGO / : / Guanine nucleotide-binding protein G(i) subunit alpha-3 / Mu-type opioid receptor / 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 speciesEscherichia coli (E. coli)
Homo sapiens (human)
Macaca mulatta (Rhesus monkey)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.05 Å
AuthorsHisano, T. / Uchikubo-Kamo, T. / Shirouzu, M. / Imai, S. / Kaneko, S. / Shimada, I.
Funding support Japan, 4items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)JP17H06097 Japan
Japan Society for the Promotion of Science (JSPS)JP21H02619 Japan
Japan Society for the Promotion of Science (JSPS)JP23KJ0574 Japan
Japan Agency for Medical Research and Development (AMED)21ae0121028h0001 Japan
CitationJournal: Nat Commun / Year: 2024
Title: Structural and dynamic insights into the activation of the μ-opioid receptor by an allosteric modulator.
Authors: Shun Kaneko / Shunsuke Imai / Tomomi Uchikubo-Kamo / Tamao Hisano / Nobuaki Asao / Mikako Shirouzu / Ichio Shimada /
Abstract: G-protein-coupled receptors (GPCRs) play pivotal roles in various physiological processes. These receptors are activated to different extents by diverse orthosteric ligands and allosteric modulators. ...G-protein-coupled receptors (GPCRs) play pivotal roles in various physiological processes. These receptors are activated to different extents by diverse orthosteric ligands and allosteric modulators. However, the mechanisms underlying these variations in signaling activity by allosteric modulators remain largely elusive. Here, we determine the three-dimensional structure of the μ-opioid receptor (MOR), a class A GPCR, in complex with the G protein and an allosteric modulator, BMS-986122, using cryogenic electron microscopy. Our results reveal that BMS-986122 binding induces changes in the map densities corresponding to R167 and Y254, key residues in the structural motifs conserved among class A GPCRs. Nuclear magnetic resonance analyses of MOR in the absence of the G protein reveal that BMS-986122 binding enhances the formation of the interaction between R167 and Y254, thus stabilizing the fully-activated conformation, where the intracellular half of TM6 is outward-shifted to allow for interaction with the G protein. These findings illuminate that allosteric modulators like BMS-986122 can potentiate receptor activation through alterations in the conformational dynamics in the core region of GPCRs. Together, our results demonstrate the regulatory mechanisms of GPCRs, providing insights into the rational development of therapeutics targeting GPCRs.
History
DepositionAug 1, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0May 29, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
R: Soluble cytochrome b562,Mu-type opioid receptor,Mu-type opioid receptor
S: DAMGO
A: G protein subunit alpha i3
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: scFv16
hetero molecules


Theoretical massNumber of molelcules
Total (without water)166,7337
Polymers166,2846
Non-polymers4491
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 2 types, 2 molecules RA

#1: Protein Soluble cytochrome b562,Mu-type opioid receptor,Mu-type opioid receptor / M-OR-1 / MOR-1 / Mu opiate receptor / Mu opioid receptor / MOP / hMOP


Mass: 51321.855 Da / Num. of mol.: 1 / Mutation: M7W,H102I,R106L,R106
Source method: isolated from a genetically manipulated source
Details: N-terminal residues (M1-G63) of MOR were replaced with the thermostabilized apocytochrome b562RIL from Escherichia coli (M7W, H102I and R106L) (BRIL) protein and a linker sequence (GSPGARSAS) ...Details: N-terminal residues (M1-G63) of MOR were replaced with the thermostabilized apocytochrome b562RIL from Escherichia coli (M7W, H102I and R106L) (BRIL) protein and a linker sequence (GSPGARSAS). C-terminal residues (Q363-P400) of MOR were truncated.,N-terminal residues (M1-G63) of MOR were replaced with the thermostabilized apocytochrome b562RIL from Escherichia coli (M7W, H102I and R106L) (BRIL) protein and a linker sequence (GSPGARSAS). C-terminal residues (Q363-P400) of MOR were truncated.
Source: (gene. exp.) Escherichia coli (E. coli), (gene. exp.) Homo sapiens (human)
Gene: cybC, OPRM1, MOR1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P35372
#3: Protein G protein subunit alpha i3 / Guanine nucleotide-binding protein G(K) subunit alpha


Mass: 40585.137 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Macaca mulatta (Rhesus monkey) / Gene: GNAI3 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: F6VL43

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

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


Mass: 38522.098 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
#5: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / G gamma-I


Mass: 7932.222 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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Protein/peptide / Antibody / Non-polymers , 3 types, 3 molecules SH

#2: Protein/peptide DAMGO


Type: Peptide-like / Class: Synthetic opioid / Mass: 513.587 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) / References: DAMGO
#6: Antibody scFv16


Mass: 27409.588 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: N-terminal (MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFA) and C-terminal (GPHHHHHHHH) residues are cleaved during purification and not present in the purified sample.
Source: (gene. exp.) synthetic construct (others) / Production host: Spodoptera frugiperda (fall armyworm)
#7: Chemical ChemComp-VV9 / (2~{S})-2-(3-bromanyl-4-methoxy-phenyl)-3-(4-chlorophenyl)sulfonyl-1,3-thiazolidine / BMS-986122


Mass: 448.782 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C16H15BrClNO3S2 / 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

ComponentName: Full agonist- and positive allosteric modulator-bound mu-type opioid receptor-G protein complex
Type: COMPLEX / Entity ID: #1-#6 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
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 FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.3 sec. / Electron dose: 50.5 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 15 eV

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Processing

EM softwareName: PHENIX / Version: 1.21_5207: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.05 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 308249 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0039112
ELECTRON MICROSCOPYf_angle_d0.55212366
ELECTRON MICROSCOPYf_dihedral_angle_d5.8691260
ELECTRON MICROSCOPYf_chiral_restr0.0411421
ELECTRON MICROSCOPYf_plane_restr0.0041549

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