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- EMDB-44635: Inactive mu opioid receptor bound to Nb6, naloxone and NAM -

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Basic information

Entry
Database: EMDB / ID: EMD-44635
TitleInactive mu opioid receptor bound to Nb6, naloxone and NAM
Map datasharpened density file for MORk/Nb6 bound to naloxone and 368
Sample
  • Complex: mu-type opioid receptor with kappa-type opioid receptor ICL3 in complex with Nb6, naloxone and a negative allosteric modulator
    • Protein or peptide: kappa opioid receptor Nanobody 6
    • Protein or peptide: Mu-type opioid receptor
  • Ligand: Naloxone
  • Ligand: Nalpha-[({(1M)-1-[5-(benzyloxy)pyridin-3-yl]naphthalen-2-yl}sulfanyl)acetyl]-3-methoxy-N,4-dimethyl-L-phenylalaninamide
KeywordsG-protein coupled receptor / inactive / opioid / allosteric modulator / MEMBRANE PROTEIN
Function / homology
Function and homology information


Opioid Signalling / spine apparatus / beta-endorphin receptor activity / morphine receptor activity / negative regulation of Wnt protein secretion / Peptide ligand-binding receptors / adenylate cyclase-inhibiting opioid receptor signaling pathway / positive regulation of appetite / G-protein activation / G protein-coupled opioid receptor activity ...Opioid Signalling / spine apparatus / beta-endorphin receptor activity / morphine receptor activity / negative regulation of Wnt protein secretion / Peptide ligand-binding receptors / adenylate cyclase-inhibiting opioid receptor signaling pathway / positive regulation of appetite / G-protein activation / G protein-coupled opioid receptor activity / negative regulation of luteinizing hormone secretion / G protein-coupled opioid receptor signaling pathway / sperm ejaculation / G alpha (i) signalling events / adenylate cyclase-inhibiting G protein-coupled acetylcholine receptor signaling pathway / negative regulation of nitric oxide biosynthetic process / negative regulation of adenylate cyclase-activating G protein-coupled receptor signaling pathway / neuropeptide binding / negative regulation of cAMP-mediated signaling / regulation of NMDA receptor activity / positive regulation of neurogenesis / eating behavior / negative regulation of cytosolic calcium ion concentration / transmission of nerve impulse / social behavior / G-protein alpha-subunit binding / GABA-ergic synapse / voltage-gated calcium channel activity / neuropeptide signaling pathway / positive regulation of gluconeogenesis / sensory perception of pain / dendrite membrane / presynaptic modulation of chemical synaptic transmission / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / excitatory postsynaptic potential / dendrite cytoplasm / locomotory behavior / G protein-coupled receptor activity / adenylate cyclase-activating dopamine receptor signaling pathway / G-protein beta-subunit binding / presynaptic membrane / phospholipase C-activating G protein-coupled receptor signaling pathway / postsynaptic membrane / perikaryon / response to ethanol / positive regulation of ERK1 and ERK2 cascade / endosome / neuron projection / G protein-coupled receptor signaling pathway / protein domain specific binding / axon / focal adhesion / dendrite / membrane / plasma membrane
Similarity search - Function
Mu opioid receptor / Opioid receptor / 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)
Similarity search - Domain/homology
Mu-type opioid receptor
Similarity search - Component
Biological speciesMus musculus (house mouse) / Lama glama (llama)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.26 Å
AuthorsO'Brien ES / Wang H / Kaavya Krishna K / Zhang C / Kobilka BK
Funding support United States, 1 items
OrganizationGrant numberCountry
Chan Zuckerberg Initiative United States
CitationJournal: Nature / Year: 2024
Title: A µ-opioid receptor modulator that works cooperatively with naloxone.
Authors: Evan S O'Brien / Vipin Ashok Rangari / Amal El Daibani / Shainnel O Eans / Haylee R Hammond / Elizabeth White / Haoqing Wang / Yuki Shiimura / Kaavya Krishna Kumar / Qianru Jiang / Kevin ...Authors: Evan S O'Brien / Vipin Ashok Rangari / Amal El Daibani / Shainnel O Eans / Haylee R Hammond / Elizabeth White / Haoqing Wang / Yuki Shiimura / Kaavya Krishna Kumar / Qianru Jiang / Kevin Appourchaux / Weijiao Huang / Chensong Zhang / Brandon J Kennedy / Jesper M Mathiesen / Tao Che / Jay P McLaughlin / Susruta Majumdar / Brian K Kobilka /
Abstract: The µ-opioid receptor (µOR) is a well-established target for analgesia, yet conventional opioid receptor agonists cause serious adverse effects, notably addiction and respiratory depression. These ...The µ-opioid receptor (µOR) is a well-established target for analgesia, yet conventional opioid receptor agonists cause serious adverse effects, notably addiction and respiratory depression. These factors have contributed to the current opioid overdose epidemic driven by fentanyl, a highly potent synthetic opioid. µOR negative allosteric modulators (NAMs) may serve as useful tools in preventing opioid overdose deaths, but promising chemical scaffolds remain elusive. Here we screened a large DNA-encoded chemical library against inactive µOR, counter-screening with active, G-protein and agonist-bound receptor to 'steer' hits towards conformationally selective modulators. We discovered a NAM compound with high and selective enrichment to inactive µOR that enhances the affinity of the key opioid overdose reversal molecule, naloxone. The NAM works cooperatively with naloxone to potently block opioid agonist signalling. Using cryogenic electron microscopy, we demonstrate that the NAM accomplishes this effect by binding a site on the extracellular vestibule in direct contact with naloxone while stabilizing a distinct inactive conformation of the extracellular portions of the second and seventh transmembrane helices. The NAM alters orthosteric ligand kinetics in therapeutically desirable ways and works cooperatively with low doses of naloxone to effectively inhibit various morphine-induced and fentanyl-induced behavioural effects in vivo while minimizing withdrawal behaviours. Our results provide detailed structural insights into the mechanism of negative allosteric modulation of the µOR and demonstrate how this can be exploited in vivo.
History
DepositionApr 25, 2024-
Header (metadata) releaseJul 17, 2024-
Map releaseJul 17, 2024-
UpdateJul 17, 2024-
Current statusJul 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44635.png

Downloads & links

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Map

FileDownload / File: emd_44635.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened density file for MORk/Nb6 bound to naloxone and 368
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.74 Å/pix.
x 300 pix.
= 222.3 Å		0.74 Å/pix.
x 300 pix.
= 222.3 Å		0.74 Å/pix.
x 300 pix.
= 222.3 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.741 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.93
Minimum - Maximum-3.6673107 - 5.0547285
Average (Standard dev.)-0.0009145511 (±0.09294792)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 222.3 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half map B for MORk/Nb6 bound to naloxone and 368

Fileemd_44635_half_map_1.map
Annotationhalf map B for MORk/Nb6 bound to naloxone and 368
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map A for MORk/Nb6 bound to naloxone and 368

Fileemd_44635_half_map_2.map
Annotationhalf map A for MORk/Nb6 bound to naloxone and 368
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : mu-type opioid receptor with kappa-type opioid receptor ICL3 in c...

EntireName: mu-type opioid receptor with kappa-type opioid receptor ICL3 in complex with Nb6, naloxone and a negative allosteric modulator
Components
  • Complex: mu-type opioid receptor with kappa-type opioid receptor ICL3 in complex with Nb6, naloxone and a negative allosteric modulator
    • Protein or peptide: kappa opioid receptor Nanobody 6
    • Protein or peptide: Mu-type opioid receptor
  • Ligand: Naloxone
  • Ligand: Nalpha-[({(1M)-1-[5-(benzyloxy)pyridin-3-yl]naphthalen-2-yl}sulfanyl)acetyl]-3-methoxy-N,4-dimethyl-L-phenylalaninamide

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Supramolecule #1: mu-type opioid receptor with kappa-type opioid receptor ICL3 in c...

SupramoleculeName: mu-type opioid receptor with kappa-type opioid receptor ICL3 in complex with Nb6, naloxone and a negative allosteric modulator
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Mus musculus (house mouse)

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Macromolecule #1: kappa opioid receptor Nanobody 6

MacromoleculeName: kappa opioid receptor Nanobody 6 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 14.730255 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MAQVQLQESG GGLVQAGESL RLSCAASGTI FRLYDMGWYR RVSGNQRELV ASITSGGSTK YGDSVKGRFT ISRDNAKNTV YLQMSSLKP EDTAVYYCNA EYRTGIWEEL LDGWGQGTQV TVSSHHHHHH EPEA

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Macromolecule #2: Mu-type opioid receptor

MacromoleculeName: Mu-type opioid receptor / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 47.920734 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MKTIIALSYI FCLVFADYKD DDDAMGPGNI SDCSDPLAPA SCSPAPGSWL NLSHVDGNQS DPCGPNRTGL GGSHSLCPQT GSPSMVTAI TIMALYSIVC VVGLFGNFLV MYVIVRYTKM KTATNIYIFN LALADALATS TLPFQSVNYL MGTWPFGNIL C KIVISIDY ...String:
MKTIIALSYI FCLVFADYKD DDDAMGPGNI SDCSDPLAPA SCSPAPGSWL NLSHVDGNQS DPCGPNRTGL GGSHSLCPQT GSPSMVTAI TIMALYSIVC VVGLFGNFLV MYVIVRYTKM KTATNIYIFN LALADALATS TLPFQSVNYL MGTWPFGNIL C KIVISIDY YNMFTSIFTL CTMSVDRYIA VCHPVKALDF RTPRNAKIVN VCNWILSSAI GLPVMFMATT KYRQGSIDCT LT FSHPTWY WENLLKICVF IFAFIMPVLI ITVCYGLMIL RLKSVRLLSG SREKDRNLRR ITRMVLVVVA VFIVCWTPIH IYV IIKALI TIPETTFQTV SWHFCIALGY TNSCLNPVLY AFLDENFKRC FREFCIPTSS TIEQQNSARI RQNTREHPST ANTV DRTNH QLENLEAETA PLPDIHHHHH H

UniProtKB: Mu-type opioid receptor

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Macromolecule #3: Naloxone

MacromoleculeName: Naloxone / type: ligand / ID: 3 / Number of copies: 1 / Formula: A1APV
Molecular weightTheoretical: 327.374 Da

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Macromolecule #4: Nalpha-[({(1M)-1-[5-(benzyloxy)pyridin-3-yl]naphthalen-2-yl}sulfa...

MacromoleculeName: Nalpha-[({(1M)-1-[5-(benzyloxy)pyridin-3-yl]naphthalen-2-yl}sulfanyl)acetyl]-3-methoxy-N,4-dimethyl-L-phenylalaninamide
type: ligand / ID: 4 / Number of copies: 1 / Formula: A1APU
Molecular weightTheoretical: 605.746 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.4000000000000001 µm / Nominal defocus min: 0.6 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7ul4

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.26 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 128613
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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