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- PDB-8qot: Structure of the mu opioid receptor bound to the antagonist nanob... -

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Basic information

Entry
Database: PDB / ID: 8qot
TitleStructure of the mu opioid receptor bound to the antagonist nanobody NbE
Components
  • Anti-Fab Nanobody
  • Mu-type opioid receptor
  • NabFab HC
  • NabFab LC
  • Nanobody E (NbE)
KeywordsMEMBRANE PROTEIN / opioid receptor / nanobody / antagonist / NabFab / MOR
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 / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / presynaptic modulation of chemical synaptic transmission / excitatory postsynaptic potential / dendrite cytoplasm / locomotory behavior / G protein-coupled receptor activity / adenylate cyclase-activating dopamine receptor signaling pathway / G-protein beta-subunit binding / phospholipase C-activating G protein-coupled receptor signaling pathway / presynaptic membrane / 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)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single 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, 3items
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, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 27, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Mu-type opioid receptor
B: Nanobody E (NbE)
H: NabFab HC
L: NabFab LC
K: Anti-Fab Nanobody


Theoretical massNumber of molelcules
Total (without water)143,8835
Polymers143,8835
Non-polymers00
Water00
1


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

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Components

#1: Protein Mu-type opioid receptor


Mass: 53994.215 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: MOR-2xSTREP-8xHIS / Source: (gene. exp.) Mus musculus (house mouse) / Gene: Oprm1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P42866
#2: Antibody Nanobody E (NbE)


Mass: 18458.662 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: synthesized nanobody NbE-6xHis / Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)
#3: Antibody NabFab HC


Mass: 28220.525 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Signal sequence-NabFab HC / Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#4: Antibody NabFab LC


Mass: 25794.859 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#5: Antibody Anti-Fab Nanobody


Mass: 17414.383 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Signal sequence-6xHis Anti-Fab nanobody / Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)

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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: MOR-NbE-NabFab-AntiFab complex / Type: COMPLEX
Details: Mu-opioid receptor bound to a nanobody antagonist (NbE), and NabFab module used as fiducial marker.
Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightValue: 0.12 MDa
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHepesC8H18N2O4S1
2100 mMSodium chlorideNaCl1
30.001 %Lauryl maltose neopentyl glycolC47H88O221
40.0001 %Cholesteryl hemisuccinateC31H50O41
SpecimenConc.: 2.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: MOR 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 ...Details: MOR 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.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 288 K

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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 FIELD / Nominal magnification: 150000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 95 K / Temperature (min): 90 K
Image recordingAverage exposure time: 52 sec. / Electron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 4 / Num. of real images: 5938
Image scansWidth: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.3.1particle selection
2EPU2.13image acquisition
4cryoSPARC3.3.1CTF correction
7UCSF Chimera1.17.1model fitting
9cryoSPARC3.3.1initial Euler assignment
10cryoSPARC3.3.1final Euler assignment
11RELION3classification
12cryoSPARC3.3.13D reconstruction
13PHENIX1.20.1model refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 6063911
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 445766 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 68 / Protocol: AB INITIO MODEL / Space: REAL
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeChain-IDInitial refinement model-ID
14DKL

4dkl

A4DKLA1
27PHQ

7phq

A7PHQA2
37PHQ

7phq

B7PHQB2
47PHQ

7phq

E7PHQE2
RefinementCross valid method: NONE

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