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- PDB-9bqj: RO76 bound muOR-Gi1-scFv16 complex structure -

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Basic information

Entry
Database: PDB / ID: 9bqj
TitleRO76 bound muOR-Gi1-scFv16 complex structure
Components
  • (Guanine nucleotide-binding protein ...) x 3
  • Mu-type opioid receptor
  • scFv16
KeywordsSIGNALING PROTEIN/IMMUNE SYSTEM / GPCR / G protein / opioid receptor / SIGNALING PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


Opioid Signalling / G-protein activation / beta-endorphin receptor activity / morphine receptor activity / negative regulation of Wnt protein secretion / Peptide ligand-binding receptors / G protein-coupled opioid receptor activity / G protein-coupled opioid receptor signaling pathway / G alpha (i) signalling events / negative regulation of nitric oxide biosynthetic process ...Opioid Signalling / G-protein activation / beta-endorphin receptor activity / morphine receptor activity / negative regulation of Wnt protein secretion / Peptide ligand-binding receptors / G protein-coupled opioid receptor activity / G protein-coupled opioid receptor signaling pathway / G alpha (i) signalling events / negative regulation of nitric oxide biosynthetic process / adenylate cyclase-inhibiting G protein-coupled acetylcholine receptor signaling pathway / regulation of NMDA receptor activity / positive regulation of neurogenesis / negative regulation of cytosolic calcium ion concentration / transmission of nerve impulse / G-protein alpha-subunit binding / adenylate cyclase inhibitor activity / positive regulation of protein localization to cell cortex / T cell migration / Adenylate cyclase inhibitory pathway / D2 dopamine receptor binding / response to prostaglandin E / adenylate cyclase regulator activity / G protein-coupled serotonin receptor binding / adenylate cyclase-inhibiting serotonin receptor signaling pathway / sensory perception of pain / presynaptic modulation of chemical synaptic transmission / cellular response to forskolin / regulation of mitotic spindle organization / Regulation of insulin secretion / locomotory behavior / positive regulation of cholesterol biosynthetic process / negative regulation of insulin secretion / G protein-coupled receptor binding / response to peptide hormone / G protein-coupled receptor activity / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / GABA-ergic synapse / centriolar satellite / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / G-protein beta/gamma-subunit complex binding / Olfactory Signaling Pathway / Activation of the phototransduction cascade / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / G protein-coupled acetylcholine receptor signaling pathway / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / G-protein activation / G beta:gamma signalling through CDC42 / Prostacyclin signalling through prostacyclin receptor / Glucagon signaling in metabolic regulation / G beta:gamma signalling through BTK / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / ADP signalling through P2Y purinoceptor 12 / photoreceptor disc membrane / Sensory perception of sweet, bitter, and umami (glutamate) taste / Glucagon-type ligand receptors / Adrenaline,noradrenaline inhibits insulin secretion / GDP binding / Vasopressin regulates renal water homeostasis via Aquaporins / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / G alpha (z) signalling events / ADP signalling through P2Y purinoceptor 1 / cellular response to catecholamine stimulus / ADORA2B mediated anti-inflammatory cytokines production / G beta:gamma signalling through PI3Kgamma / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / adenylate cyclase-activating dopamine receptor signaling pathway / GPER1 signaling / Inactivation, recovery and regulation of the phototransduction cascade / G-protein beta-subunit binding / cellular response to prostaglandin E stimulus / heterotrimeric G-protein complex / G alpha (12/13) signalling events / presynapse / extracellular vesicle / sensory perception of taste / Thrombin signalling through proteinase activated receptors (PARs) / signaling receptor complex adaptor activity / retina development in camera-type eye / G protein activity / GTPase binding / Ca2+ pathway / fibroblast proliferation / midbody / cell cortex / High laminar flow shear stress activates signaling by PIEZO1 and PECAM1:CDH5:KDR in endothelial cells / G alpha (i) signalling events / G alpha (s) signalling events / phospholipase C-activating G protein-coupled receptor signaling pathway / perikaryon / G alpha (q) signalling events / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / Ras protein signal transduction / Extra-nuclear estrogen signaling / positive regulation of ERK1 and ERK2 cascade / cell population proliferation / endosome
Similarity search - Function
Mu opioid receptor / Opioid receptor / G-protein alpha subunit, group I / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G-alpha domain profile. / 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 / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G-alpha domain profile. / G protein alpha subunit / G-protein, gamma subunit / G-protein gamma subunit domain profile. / G-protein gamma-like domain / G-protein gamma-like domain superfamily / GGL domain / G protein gamma subunit-like motifs / GGL domain / G protein beta WD-40 repeat protein / 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. / 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
: / 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 / Guanine nucleotide-binding protein G(i) subunit alpha-1
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsWang, H. / Majumdar, S. / Kobilka, B.K.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute on Drug Abuse (NIH/NIDA)DA057790 United States
CitationJournal: ACS Cent Sci / Year: 2024
Title: Signaling Modulation Mediated by Ligand Water Interactions with the Sodium Site at μOR.
Authors: Rohini S Ople / Nokomis Ramos-Gonzalez / Qiongyu Li / Briana L Sobecks / Deniz Aydin / Alexander S Powers / Abdelfattah Faouzi / Benjamin J Polacco / Sarah M Bernhard / Kevin Appourchaux / ...Authors: Rohini S Ople / Nokomis Ramos-Gonzalez / Qiongyu Li / Briana L Sobecks / Deniz Aydin / Alexander S Powers / Abdelfattah Faouzi / Benjamin J Polacco / Sarah M Bernhard / Kevin Appourchaux / Sashrik Sribhashyam / Shainnel O Eans / Bowen A Tsai / Ron O Dror / Balazs R Varga / Haoqing Wang / Ruth Hüttenhain / Jay P McLaughlin / Susruta Majumdar /
Abstract: The mu opioid receptor (μOR) is a target for clinically used analgesics. However, adverse effects, such as respiratory depression and physical dependence, necessitate the development of alternative ...The mu opioid receptor (μOR) is a target for clinically used analgesics. However, adverse effects, such as respiratory depression and physical dependence, necessitate the development of alternative treatments. Recently we reported a novel strategy to design functionally selective opioids by targeting the sodium binding allosteric site in μOR with a supraspinally active analgesic named . Presently, to improve systemic activity of this ligand, we used structure-based design, identifying a new ligand named where the flexible alkyl linker and polar guanidine guano group is swapped with a benzyl alcohol, and the sodium site is targeted indirectly through waters. A cryoEM structure of bound to the μOR-G complex confirmed that interacts with the sodium site residues through a water molecule, unlike which engages the sodium site directly. Signaling assays coupled with APEX based proximity labeling show binding in the sodium pocket modulates receptor efficacy and trafficking. In mice, was systemically active in tail withdrawal assays and showed reduced liabilities compared to those of morphine. In summary, we show that targeting water molecules in the sodium binding pocket may be an avenue to modulate signaling properties of opioids, and which may potentially be extended to other G-protein coupled receptors where this site is conserved.
History
DepositionMay 10, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 11, 2024Provider: repository / Type: Initial release
Revision 1.0Sep 11, 2024Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Sep 11, 2024Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Sep 11, 2024Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Sep 11, 2024Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Sep 11, 2024Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Sep 11, 2024Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Sep 11, 2024Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Sep 11, 2024Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Sep 11, 2024Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Oct 9, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification
Revision 1.2May 21, 2025Group: Data collection / Category: em_admin / em_software / Item: _em_admin.last_update / _em_software.name
Revision 1.1May 21, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
C: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
D: Mu-type opioid receptor
A: Guanine nucleotide-binding protein G(i) subunit alpha-1
E: scFv16
hetero molecules


Theoretical massNumber of molelcules
Total (without water)154,1086
Polymers153,7275
Non-polymers3811
Water181
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Guanine nucleotide-binding protein ... , 3 types, 3 molecules BCA

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


Mass: 37671.102 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNB1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P62873
#2: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / G gamma-I


Mass: 7861.143 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
#4: Protein Guanine nucleotide-binding protein G(i) subunit alpha-1 / Adenylate cyclase-inhibiting G alpha protein


Mass: 40415.031 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNAI1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P63096

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Protein / Antibody , 2 types, 2 molecules DE

#3: Protein Mu-type opioid receptor / M-OR-1 / MOR-1


Mass: 39995.105 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Oprm1, Mor, Oprm / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P42866
#5: Antibody scFv16


Mass: 27784.896 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Trichoplusia ni (cabbage looper)

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Non-polymers , 2 types, 2 molecules

#6: Chemical ChemComp-A1AQ2 / N-{[3-(hydroxymethyl)phenyl]methyl}-N-[1-(2-phenylethyl)piperidin-4-yl]propanamide


Mass: 380.523 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C24H32N2O2 / Feature type: SUBJECT OF INVESTIGATION
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationY

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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: RO76 bound muOR-Gi1-scFv16 complex / Type: COMPLEX / Entity ID: #1-#5 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7
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: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 700 nm / C2 aperture diameter: 50 µm
Image recordingElectron dose: 54.45 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2578269
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 127261 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0028699
ELECTRON MICROSCOPYf_angle_d0.48811817
ELECTRON MICROSCOPYf_dihedral_angle_d9.4431221
ELECTRON MICROSCOPYf_chiral_restr0.041377
ELECTRON MICROSCOPYf_plane_restr0.0031483

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