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- EMDB-18541: Structure of the mu opioid receptor bound to the antagonist nanob... -

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Basic information

Entry
Database: EMDB / ID: EMD-18541
TitleStructure of the mu opioid receptor bound to the antagonist nanobody NbE
Map dataDeepEMhancer sharpened map of the MOR_NbE_NabFab_anti-Fab complex.
Sample
  • Complex: MOR-NbE-NabFab-AntiFab complex
    • Protein or peptide: Mu-type opioid receptor
    • Protein or peptide: Nanobody E (NbE)
    • Protein or peptide: NabFab HC
    • Protein or peptide: NabFab LC
    • Protein or peptide: Anti-Fab Nanobody
Keywordsopioid receptor / nanobody / antagonist / NabFab / MOR / 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 cAMP-mediated signaling / negative regulation of adenylate cyclase-activating G protein-coupled receptor signaling pathway / neuropeptide binding / regulation of NMDA receptor activity / positive regulation of neurogenesis / eating behavior / negative regulation of cytosolic calcium ion concentration / transmission of nerve impulse / social behavior / neuropeptide signaling pathway / G-protein alpha-subunit binding / voltage-gated calcium channel activity / GABA-ergic synapse / positive regulation of gluconeogenesis / dendrite membrane / sensory perception of pain / presynaptic modulation of chemical synaptic transmission / dendrite cytoplasm / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / excitatory postsynaptic potential / 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 / perikaryon / postsynaptic membrane / 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) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsYu J / Kumar A / Zhang X / Martin C / Raia P / Manglik A / Ballet S / Boland A / Stoeber M
Funding support Switzerland, 3 items
OrganizationGrant numberCountry
Swiss National Science FoundationTMSGI3_211581 Switzerland
Swiss National Science Foundation310030_185235 Switzerland
Swiss National Science FoundationPCEFP3_181282 Switzerland
CitationJournal: bioRxiv / Year: 2023
Title: Structural Basis of μ-Opioid Receptor-Targeting by a Nanobody Antagonist.
Authors: Jun Yu / Amit Kumar / Xuefeng Zhang / Charlotte Martin / Pierre Raia / Antoine Koehl / Toon Laeremans / Jan Steyaert / Aashish Manglik / Steven Ballet / Andreas Boland / Miriam Stoeber /
Abstract: The μ-opioid receptor (μOR), a prototypical member of the G protein-coupled receptor (GPCR) family, is the molecular target of opioid analgesics such as morphine and fentanyl. Due to the ...The μ-opioid receptor (μOR), a prototypical member of the G protein-coupled receptor (GPCR) family, is the molecular target of opioid analgesics such as morphine and fentanyl. Due to the limitations and severe side effects of currently available opioid drugs, there is considerable interest in developing novel modulators of μOR function. Most GPCR ligands today are small molecules, however biologics, including antibodies and nanobodies, are emerging as alternative therapeutics with clear advantages such as affinity and target selectivity. Here, we describe the nanobody NbE, which selectively binds to the μOR and acts as an antagonist. We functionally characterize NbE as an extracellular and genetically encoded μOR ligand and uncover the molecular basis for μOR antagonism by solving the cryo-EM structure of the NbE-μOR complex. NbE displays a unique ligand binding mode and achieves μOR selectivity by interactions with the orthosteric pocket and extracellular receptor loops. Based on a β-hairpin loop formed by NbE that deeply inserts into the μOR and centers most binding contacts, we design short peptide analogues that retain μOR antagonism. The work illustrates the potential of nanobodies to uniquely engage with GPCRs and describes novel μOR ligands that can serve as a basis for therapeutic developments.
History
DepositionSep 29, 2023-
Header (metadata) releaseDec 27, 2023-
Map releaseDec 27, 2023-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18541.png

Downloads & links

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Map

FileDownload / File: emd_18541.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDeepEMhancer sharpened map of the MOR_NbE_NabFab_anti-Fab complex.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.98 Å/pix.
x 320 pix.
= 312.288 Å		0.98 Å/pix.
x 320 pix.
= 312.288 Å		0.98 Å/pix.
x 320 pix.
= 312.288 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.9759 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.044121403 - 1.7903484
Average (Standard dev.)0.00039334194 (±0.016585164)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 312.288 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_18541_msk_1.map
Projections & Slices
AxesZYX

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Additional map: Unsharpened map of the MOR NbE NabFab anti-Fab complex.

Fileemd_18541_additional_1.map
AnnotationUnsharpened map of the MOR_NbE_NabFab_anti-Fab complex.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Additional map: Unsharpened MOR NbE map (focused refinement).

Fileemd_18541_additional_2.map
AnnotationUnsharpened MOR_NbE map (focused refinement).
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Additional map: DeepEMhancer sharpened MOR NbE map (focused refinement).

Fileemd_18541_additional_3.map
AnnotationDeepEMhancer sharpened MOR_NbE map (focused refinement).
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Half map A of the MOR NbE map (focused refinement).

Fileemd_18541_additional_4.map
AnnotationHalf map A of the MOR_NbE map (focused refinement).
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Additional map: Half map B of the MOR NbE map (focused refinement).

Fileemd_18541_additional_5.map
AnnotationHalf map B of the MOR_NbE map (focused refinement).
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A of the MOR NbE NabFab anti-Fab complex.

Fileemd_18541_half_map_1.map
AnnotationHalf map A of the MOR_NbE_NabFab_anti-Fab complex.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B of the MOR NbE NabFab anti-Fab complex.

Fileemd_18541_half_map_2.map
AnnotationHalf map B of the MOR_NbE_NabFab_anti-Fab complex.
Projections & Slices
AxesZYX

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Sample components

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Entire : MOR-NbE-NabFab-AntiFab complex

EntireName: MOR-NbE-NabFab-AntiFab complex
Components
  • Complex: MOR-NbE-NabFab-AntiFab complex
    • Protein or peptide: Mu-type opioid receptor
    • Protein or peptide: Nanobody E (NbE)
    • Protein or peptide: NabFab HC
    • Protein or peptide: NabFab LC
    • Protein or peptide: Anti-Fab Nanobody

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Supramolecule #1: MOR-NbE-NabFab-AntiFab complex

SupramoleculeName: MOR-NbE-NabFab-AntiFab complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Mu-opioid receptor bound to a nanobody antagonist (NbE), and NabFab module used as fiducial marker.
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 120 KDa

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Macromolecule #1: Mu-type opioid receptor

MacromoleculeName: Mu-type opioid receptor / type: protein_or_peptide / ID: 1 / Details: MOR-2xSTREP-8xHIS / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 53.994215 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MKTIIALSYI FCLVFADYKD DDDAMGPGNI SDCSDPLAPA SCSPAPGSWL NLSHVDGNQS DPCGPNRTGL GENLYFQGSH SLCPQTGSP SMVTAITIMA LYSIVCVVGL FGNFLVMYVI VRYTKMKTAT NIYIFNLALA DALATSTLPF QSVNYLMGTW P FGNILCKI ...String:
MKTIIALSYI FCLVFADYKD DDDAMGPGNI SDCSDPLAPA SCSPAPGSWL NLSHVDGNQS DPCGPNRTGL GENLYFQGSH SLCPQTGSP SMVTAITIMA LYSIVCVVGL FGNFLVMYVI VRYTKMKTAT NIYIFNLALA DALATSTLPF QSVNYLMGTW P FGNILCKI VISIDYYNMF TSIFTLCTMS VDRYIAVCHP VKALDFRTPR NAKIVNVCNW ILSSAIGLPV MFMATTKYRQ GS IDCTLTF SHPTWYWENL LKICVFIFAF IMPVLIITVC YGLMILRLKS VRMLSGSKEK DRNLRRITRM VLVVVAVFIV CWT PIHIYV IIKALITIPE TTFQTVSWHF CIALGYTNSC LNPVLYAFLD ENFKRCFREF CIPTSSTILE VLFQGPEQQN SARI RQNTR EHPSTANTVD RTNHQLENLE AETAPLPSSL AEENLYFQSW SHPQFEKGGG SGGGSGGGSW SHPQFEKGAH HHHHH HH

UniProtKB: Mu-type opioid receptor

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Macromolecule #2: Nanobody E (NbE)

MacromoleculeName: Nanobody E (NbE) / type: protein_or_peptide / ID: 2 / Details: synthesized nanobody NbE-6xHis / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 18.458662 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
VKKLLFAIPL VVPFYAAQPA MAQVQLVESG GGLVQAGGSL RLSCAASGSI SSISTMGWYR QAPGKERELV AAITSGGSTN YADSVKGRF TISRDNAKNT VYLQMNSLKP EDTAVYYCNF KYYSGSYFYK SEYDYWGKGT PVTVSSAAAH HHHHHGAAEQ K LISEEDLN GAA

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Macromolecule #3: NabFab HC

MacromoleculeName: NabFab HC / type: protein_or_peptide / ID: 3 / Details: Signal sequence-NabFab HC / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 28.220525 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKKNIAFLLA SMFVFSIATN AYAEISEVQL VESGGGLVQP GGSLRLSCAA SGFNFSYYSI HWVRQAPGKG LEWVAYISSS SSYTSYADS VKGRFTISAD TSKNTAYLQM NSLRAEDTAV YYCARGYQYW QYHASWYWNG GLDYWGQGTL VTVSSASTKG P SVFPLAPS ...String:
MKKNIAFLLA SMFVFSIATN AYAEISEVQL VESGGGLVQP GGSLRLSCAA SGFNFSYYSI HWVRQAPGKG LEWVAYISSS SSYTSYADS VKGRFTISAD TSKNTAYLQM NSLRAEDTAV YYCARGYQYW QYHASWYWNG GLDYWGQGTL VTVSSASTKG P SVFPLAPS SKSTSGGTAA LGCLVKDYFP EPVTVSWNSG ALTSGVHTFP AVLQSSGLYS LSSVVTVPSS SLGTQTYICN VN HKPSNTK VDKKVEPKSC DKTHT

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Macromolecule #4: NabFab LC

MacromoleculeName: NabFab LC / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 25.794859 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKKNIAFLLA SMFVFSIATN AYASDIQMTQ SPSSLSASVG DRVTITCRAS QSVSSAVAWY QQKPGKAPKL LIYSASSLYS GVPSRFSGS RSGTDFTLTI SSLQPEDFAT YYCQQSSSSL ITFGQGTKVE IKRTVAAPSV FIFPPSDSQL KSGTASVVCL L NNFYPREA ...String:
MKKNIAFLLA SMFVFSIATN AYASDIQMTQ SPSSLSASVG DRVTITCRAS QSVSSAVAWY QQKPGKAPKL LIYSASSLYS GVPSRFSGS RSGTDFTLTI SSLQPEDFAT YYCQQSSSSL ITFGQGTKVE IKRTVAAPSV FIFPPSDSQL KSGTASVVCL L NNFYPREA KVQWKVDNAL QSGNSQESVT EQDSKDSTYS LSSTLTLSKA DYEKHKVYAC EVTHQGLSSP VTKSFNRGEC

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Macromolecule #5: Anti-Fab Nanobody

MacromoleculeName: Anti-Fab Nanobody / type: protein_or_peptide / ID: 5 / Details: Signal sequence-6xHis Anti-Fab nanobody / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 17.414383 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MKYLLPTAAA GLLLLAAQPA MAMHHHHHHG ENLYFQGSQV QLQESGGGLV QPGGSLRLSC AASGRTISRY AMSWFRQAPG KEREFVAVA RRSGDGAFYA DSVQGRFTVS RDDAKNTVYL QMNSLKPEDT AVYYCAIDSD TFYSGSYDYW GQGTQVTVSS

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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

Concentration2.8 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHepes
100.0 mMNaClSodium chloride
0.001 %C47H88O22Lauryl maltose neopentyl glycol
0.0001 %C31H50O4Cholesteryl hemisuccinate
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 288 K / Instrument: LEICA EM GP
DetailsMOR receptor was expressed and purified in insect cells in isolation. NbE was expressed and purified in E. coli. NabFab module was expressed and purified in E. coli. All purified components were incubated and formed readily a stable complex. Final complex was purified over size exclusion chromatography.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
TemperatureMin: 90.0 K / Max: 95.0 K
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 4 / Number real images: 5938 / Average exposure time: 52.0 sec. / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 150000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 6063911
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.1) / Number images used: 445766
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1)
Final 3D classificationSoftware - Name: RELION (ver. 3)

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Atomic model buiding 1

Initial model
PDB IDChain

4dkl

chain_id: A, source_name: PDB, initial_model_type: experimental model

7phq

chain_id: A, source_name: PDB, initial_model_type: experimental model

7phq

chain_id: B, source_name: PDB, initial_model_type: experimental model

7phq

chain_id: E, source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 68
Output model

PDB-8qot:
Structure of the mu opioid receptor bound to the antagonist nanobody NbE

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