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- PDB-7l1v: Orexin Receptor 2 (OX2R) in Complex with G Protein and Small-Mole... -

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Basic information

Entry
Database: PDB / ID: 7l1v
TitleOrexin Receptor 2 (OX2R) in Complex with G Protein and Small-Molecule Agonist Compound 1
Components
  • (Guanine nucleotide-binding protein ...) x 2
  • Engineered Guanine nucleotide-binding protein subunit alpha
  • Hypocretin receptor type 2
  • Synthetic nanobody 51 (Sb51)
  • single-chain antibody Fv fragment (svFv16)
KeywordsMEMBRANE PROTEIN / Class A GPCR / Orexin receptor 2 / OX2R / Peptide agonist
Function / homology
Function and homology information


regulation of circadian sleep/wake cycle, wakefulness / circadian sleep/wake cycle process / orexin receptor activity / Orexin and neuropeptides FF and QRFP bind to their respective receptors / neuropeptide receptor activity / locomotion / feeding behavior / peptide hormone binding / neuropeptide signaling pathway / regulation of cytosolic calcium ion concentration ...regulation of circadian sleep/wake cycle, wakefulness / circadian sleep/wake cycle process / orexin receptor activity / Orexin and neuropeptides FF and QRFP bind to their respective receptors / neuropeptide receptor activity / locomotion / feeding behavior / peptide hormone binding / neuropeptide signaling pathway / regulation of cytosolic calcium ion concentration / cellular response to hormone stimulus / 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 / Prostacyclin signalling through prostacyclin receptor / G beta:gamma signalling through CDC42 / 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 / Vasopressin regulates renal water homeostasis via Aquaporins / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / G alpha (z) signalling events / cellular response to catecholamine stimulus / ADP signalling through P2Y purinoceptor 1 / ADORA2B mediated anti-inflammatory cytokines production / G beta:gamma signalling through PI3Kgamma / adenylate cyclase-activating dopamine receptor signaling pathway / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / GPER1 signaling / Inactivation, recovery and regulation of the phototransduction cascade / cellular response to prostaglandin E stimulus / G-protein beta-subunit binding / heterotrimeric G-protein complex / G alpha (12/13) signalling events / sensory perception of taste / extracellular vesicle / signaling receptor complex adaptor activity / Thrombin signalling through proteinase activated receptors (PARs) / retina development in camera-type eye / GTPase binding / Ca2+ pathway / fibroblast proliferation / 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 / G alpha (q) signalling events / chemical synaptic transmission / Ras protein signal transduction / Extra-nuclear estrogen signaling / cell population proliferation / G protein-coupled receptor signaling pathway / lysosomal membrane / GTPase activity / synapse / protein-containing complex binding / signal transduction / extracellular exosome / nucleoplasm / membrane / plasma membrane / cytoplasm / cytosol
Similarity search - Function
Orexin receptor 2 / Orexin receptor type 2 / Orexin receptor family / YVTN repeat-like/Quinoprotein amine dehydrogenase / Serpentine type 7TM GPCR chemoreceptor Srsx / 7 Propeller / Methylamine Dehydrogenase; Chain H / G-protein, gamma subunit / G-protein gamma subunit domain profile. / G-protein gamma-like domain ...Orexin receptor 2 / Orexin receptor type 2 / Orexin receptor family / YVTN repeat-like/Quinoprotein amine dehydrogenase / Serpentine type 7TM GPCR chemoreceptor Srsx / 7 Propeller / Methylamine Dehydrogenase; Chain H / 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) / WD domain, G-beta repeat / G-protein beta WD-40 repeat / P-loop containing nucleotide triphosphate hydrolases / 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 / Immunoglobulins / Immunoglobulin-like / Sandwich / Rossmann fold / 3-Layer(aba) Sandwich / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
Chem-XGD / Orexin receptor type 2 / 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 / Orexin receptor type 2
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsHong, C. / Byrne, N.J. / Zamlynny, B. / Tummala, S. / Xiao, L. / Shipman, J.M. / Partridge, A.T. / Minnick, C. / Breslin, M.J. / Rudd, M.T. ...Hong, C. / Byrne, N.J. / Zamlynny, B. / Tummala, S. / Xiao, L. / Shipman, J.M. / Partridge, A.T. / Minnick, C. / Breslin, M.J. / Rudd, M.T. / Stachel, S.J. / Rada, V.L. / Kern, J.C. / Armacost, K.A. / Hollingsworth, S.A. / O'Brien, J.A. / Hall, D.L. / McDonald, T.P. / Strickland, C. / Brooun, A. / Soisson, S.M. / Hollenstein, K.
CitationJournal: Nat Commun / Year: 2021
Title: Structures of active-state orexin receptor 2 rationalize peptide and small-molecule agonist recognition and receptor activation.
Authors: Chuan Hong / Noel J Byrne / Beata Zamlynny / Srivanya Tummala / Li Xiao / Jennifer M Shipman / Andrea T Partridge / Christina Minnick / Michael J Breslin / Michael T Rudd / Shawn J Stachel / ...Authors: Chuan Hong / Noel J Byrne / Beata Zamlynny / Srivanya Tummala / Li Xiao / Jennifer M Shipman / Andrea T Partridge / Christina Minnick / Michael J Breslin / Michael T Rudd / Shawn J Stachel / Vanessa L Rada / Jeffrey C Kern / Kira A Armacost / Scott A Hollingsworth / Julie A O'Brien / Dawn L Hall / Terrence P McDonald / Corey Strickland / Alexei Brooun / Stephen M Soisson / Kaspar Hollenstein /
Abstract: Narcolepsy type 1 (NT1) is a chronic neurological disorder that impairs the brain's ability to control sleep-wake cycles. Current therapies are limited to the management of symptoms with modest ...Narcolepsy type 1 (NT1) is a chronic neurological disorder that impairs the brain's ability to control sleep-wake cycles. Current therapies are limited to the management of symptoms with modest effectiveness and substantial adverse effects. Agonists of the orexin receptor 2 (OXR) have shown promise as novel therapeutics that directly target the pathophysiology of the disease. However, identification of drug-like OXR agonists has proven difficult. Here we report cryo-electron microscopy structures of active-state OXR bound to an endogenous peptide agonist and a small-molecule agonist. The extended carboxy-terminal segment of the peptide reaches into the core of OXR to stabilize an active conformation, while the small-molecule agonist binds deep inside the orthosteric pocket, making similar key interactions. Comparison with antagonist-bound OXR suggests a molecular mechanism that rationalizes both receptor activation and inhibition. Our results enable structure-based discovery of therapeutic orexin agonists for the treatment of NT1 and other hypersomnia disorders.
History
DepositionDec 15, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 10, 2021Provider: repository / Type: Initial release
Revision 1.1Feb 17, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Oct 30, 2024Group: Data collection / Database references ...Data collection / Database references / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Movie
  • Deposited structure unit
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Engineered Guanine nucleotide-binding protein subunit alpha
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
H: single-chain antibody Fv fragment (svFv16)
R: Hypocretin receptor type 2
S: Synthetic nanobody 51 (Sb51)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)157,9587
Polymers157,3336
Non-polymers6251
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 3 types, 3 molecules ARS

#1: Protein Engineered Guanine nucleotide-binding protein subunit alpha


Mass: 28040.881 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#5: Protein Hypocretin receptor type 2 / Orexin receptor type 2


Mass: 43470.875 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HCRTR2 / Production host: Homo sapiens (human) / References: UniProt: Q548Y0, UniProt: O43614*PLUS
#6: Protein Synthetic nanobody 51 (Sb51)


Mass: 12786.225 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)

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

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


Mass: 38579.148 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
#3: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / G gamma-I


Mass: 7845.078 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

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Antibody / Non-polymers , 2 types, 2 molecules H

#4: Antibody single-chain antibody Fv fragment (svFv16)


Mass: 26610.615 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Escherichia coli (E. coli)
#7: Chemical ChemComp-XGD / 4'-methoxy-N,N-dimethyl-3'-{[3-(2-{[2-(2H-1,2,3-triazol-2-yl)benzene-1-carbonyl]amino}ethyl)phenyl]sulfamoyl}[1,1'-biphenyl]-3-carboxamide


Mass: 624.709 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C33H32N6O5S / Feature type: SUBJECT OF INVESTIGATION

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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: Orexin Receptor 2 (OX2R) in Complex with G Protein and Small-Molecule Agonist Compound 1
Type: COMPLEX / Entity ID: #1-#6 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Homo sapiens (human)9606
21Mus musculus (house mouse)10090
31Lama glama (llama)9844
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
11Escherichia coli (E. coli)562
21Trichoplusia ni (cabbage looper)7111
31Homo sapiens (human)9606
Buffer solutionpH: 7.6
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTrisTris1
250 mMammonium acetateNH4OAc1
30.02 percentDDMDDM1
40.002 percentCHSCHS1
50.5 mMEDTAEDTA1
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Carbon side facing up / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K / Details: 3s blot from both sides

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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 / Calibrated magnification: 59524 X / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm / Calibrated defocus min: 600 nm / Calibrated defocus max: 2200 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 93 K / Temperature (min): 83 K
Image recordingAverage exposure time: 0.05 sec. / Electron dose: 1.0625 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 18000 / Details: 40 frames with total 2s exposure per movie
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansSampling size: 5 µm / Width: 5700 / Height: 4100

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC2.15particle selection
2Latitude3.32.2403image acquisitionGatan
4cryoSPARC2.15CTF correction
7Coot0.8.9.2model fitting
9cryoSPARC2.15initial Euler assignment
10cryoSPARC2.15final Euler assignment
11cryoSPARC2.15classification
12cryoSPARC2.153D reconstruction
13PHENIX1.15model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 7500000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1100000 / Algorithm: FOURIER SPACE / Details: Gold standard with masking effect correction / Num. of class averages: 1 / Symmetry type: POINT
Atomic model building
IDB valueProtocolSpaceTarget criteria
1101AB INITIO MODELREALCorrelation
2
3
4
5
Atomic model building

Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-ID 3D fitting-IDAccession codeDetailsInitial refinement model-ID
15G53

5g53

C15G53chain A1
23SN6

3sn6

B23SN6chain B2
33SN6

3sn6

G33SN6chain C2
46DDE

6dde

E46DDEchain H3
54S0V

4s0v

A54S0Vchain R4

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