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- PDB-8gus: Cryo-EM structure of HU-CB2-G protein complex -

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Basic information

Entry
Database: PDB / ID: 8gus
TitleCryo-EM structure of HU-CB2-G protein complex
Components
  • (Guanine nucleotide-binding protein ...) x 3
  • Cannabinoid receptor 2
  • scFv16
KeywordsSTRUCTURAL PROTEIN / GPCR / G protein / cryo-EM / membrane protein
Function / homology
Function and homology information


cannabinoid receptor activity / negative regulation of mast cell activation / negative regulation of synaptic transmission, GABAergic / negative regulation of action potential / Class A/1 (Rhodopsin-like receptors) / regulation of metabolic process / leukocyte chemotaxis / extrinsic component of cytoplasmic side of plasma membrane / G protein-coupled receptor signaling pathway, coupled to cyclic nucleotide second messenger / T cell migration ...cannabinoid receptor activity / negative regulation of mast cell activation / negative regulation of synaptic transmission, GABAergic / negative regulation of action potential / Class A/1 (Rhodopsin-like receptors) / regulation of metabolic process / leukocyte chemotaxis / extrinsic component of cytoplasmic side of plasma membrane / G protein-coupled receptor signaling pathway, coupled to cyclic nucleotide second messenger / T cell migration / Adenylate cyclase inhibitory pathway / positive regulation of protein localization to cell cortex / regulation of cAMP-mediated signaling / D2 dopamine receptor binding / G protein-coupled serotonin receptor binding / regulation of mitotic spindle organization / cellular response to forskolin / response to amphetamine / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / Regulation of insulin secretion / G protein-coupled receptor binding / G-protein beta/gamma-subunit complex binding / adenylate cyclase-activating G protein-coupled receptor signaling pathway / Olfactory Signaling Pathway / Activation of the phototransduction cascade / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / G protein-coupled acetylcholine receptor signaling pathway / G-protein activation / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / Prostacyclin signalling through prostacyclin receptor / response to peptide hormone / Glucagon signaling in metabolic regulation / G beta:gamma signalling through CDC42 / G beta:gamma signalling through BTK / ADP signalling through P2Y purinoceptor 12 / Sensory perception of sweet, bitter, and umami (glutamate) taste / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / photoreceptor disc membrane / Glucagon-type ligand receptors / Adrenaline,noradrenaline inhibits insulin secretion / Vasopressin regulates renal water homeostasis via Aquaporins / G alpha (z) signalling events / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / cellular response to catecholamine stimulus / ADORA2B mediated anti-inflammatory cytokines production / sensory perception of taste / ADP signalling through P2Y purinoceptor 1 / 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 / GDP binding / cellular response to prostaglandin E stimulus / Inactivation, recovery and regulation of the phototransduction cascade / G-protein beta-subunit binding / heterotrimeric G-protein complex / G alpha (12/13) signalling events / extracellular vesicle / signaling receptor complex adaptor activity / Thrombin signalling through proteinase activated receptors (PARs) / GTPase binding / retina development in camera-type eye / Ca2+ pathway / phospholipase C-activating G protein-coupled receptor signaling pathway / cell cortex / midbody / G alpha (i) signalling events / fibroblast proliferation / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / G alpha (s) signalling events / G alpha (q) signalling events / perikaryon / Ras protein signal transduction / response to lipopolysaccharide / cell population proliferation / Extra-nuclear estrogen signaling / inflammatory response / immune response / G protein-coupled receptor signaling pathway / cell division / lysosomal membrane / GTPase activity / centrosome / dendrite / synapse / protein-containing complex binding / nucleolus / GTP binding / magnesium ion binding / signal transduction / endoplasmic reticulum / extracellular exosome / nucleoplasm / membrane / plasma membrane / cytosol
Similarity search - Function
Cannabinoid receptor type 2 / Cannabinoid receptor family / G-protein alpha subunit, group I / Serpentine type 7TM GPCR chemoreceptor Srsx / 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 ...Cannabinoid receptor type 2 / Cannabinoid receptor family / G-protein alpha subunit, group I / Serpentine type 7TM GPCR chemoreceptor Srsx / 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 / 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. / WD domain, G-beta repeat / 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
Chem-KO3 / Cannabinoid receptor 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 / Guanine nucleotide-binding protein G(i) subunit alpha-1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.97 Å
AuthorsWu, L.J. / Hua, T. / Liu, Z.J. / Li, X.T. / Chang, H.
Funding support China, 2items
OrganizationGrant numberCountry
National Science Foundation (NSF, China)32022038 China
National Natural Science Foundation of China (NSFC)31870744 China
CitationJournal: Nat Commun / Year: 2023
Title: Structural basis of selective cannabinoid CB receptor activation.
Authors: Xiaoting Li / Hao Chang / Jara Bouma / Laura V de Paus / Partha Mukhopadhyay / Janos Paloczi / Mohammed Mustafa / Cas van der Horst / Sanjay Sunil Kumar / Lijie Wu / Yanan Yu / Richard J B H ...Authors: Xiaoting Li / Hao Chang / Jara Bouma / Laura V de Paus / Partha Mukhopadhyay / Janos Paloczi / Mohammed Mustafa / Cas van der Horst / Sanjay Sunil Kumar / Lijie Wu / Yanan Yu / Richard J B H N van den Berg / Antonius P A Janssen / Aron Lichtman / Zhi-Jie Liu / Pal Pacher / Mario van der Stelt / Laura H Heitman / Tian Hua /
Abstract: Cannabinoid CB receptor (CBR) agonists are investigated as therapeutic agents in the clinic. However, their molecular mode-of-action is not fully understood. Here, we report the discovery of LEI-102, ...Cannabinoid CB receptor (CBR) agonists are investigated as therapeutic agents in the clinic. However, their molecular mode-of-action is not fully understood. Here, we report the discovery of LEI-102, a CBR agonist, used in conjunction with three other CBR ligands (APD371, HU308, and CP55,940) to investigate the selective CBR activation by binding kinetics, site-directed mutagenesis, and cryo-EM studies. We identify key residues for CBR activation. Highly lipophilic HU308 and the endocannabinoids, but not the more polar LEI-102, APD371, and CP55,940, reach the binding pocket through a membrane channel in TM1-TM7. Favorable physico-chemical properties of LEI-102 enable oral efficacy in a chemotherapy-induced nephropathy model. This study delineates the molecular mechanism of CBR activation by selective agonists and highlights the role of lipophilicity in CBR engagement. This may have implications for GPCR drug design and sheds light on their activation by endogenous ligands.
History
DepositionSep 13, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0May 10, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 6, 2024Group: Data collection / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._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

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Guanine nucleotide-binding protein G(i) subunit alpha-1
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
R: Cannabinoid receptor 2
S: scFv16
hetero molecules


Theoretical massNumber of molelcules
Total (without water)153,6156
Polymers153,2015
Non-polymers4151
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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

#1: 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: Spodoptera frugiperda (fall armyworm) / References: UniProt: P63096
#2: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Transducin beta chain 1 / G protein beta subunit


Mass: 37416.930 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNB1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P62873
#3: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / G gamma-I / G protein gamma subunit


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: Spodoptera frugiperda (fall armyworm) / References: UniProt: P59768

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Protein / Antibody / Non-polymers , 3 types, 3 molecules RS

#4: Protein Cannabinoid receptor 2 / hCB2 / CX5


Mass: 39722.715 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CNR2, CB2A, CB2B / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P34972
#5: Antibody scFv16


Mass: 27784.896 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Spodoptera frugiperda (fall armyworm)
#6: Chemical ChemComp-KO3 / [(1~{S},4~{S},5~{S})-4-[2,6-dimethoxy-4-(2-methyloctan-2-yl)phenyl]-6,6-dimethyl-2-bicyclo[3.1.1]hept-2-enyl]methanol


Mass: 414.621 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C27H42O3 / 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: Ternary complex of Cannabinoid receptor 2 with Guanine nucleotide-binding protein and Single-chain variable fragment
Type: COMPLEX / Entity ID: #1-#5 / Source: RECOMBINANT
Molecular weightValue: 140 kDa/nm / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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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 / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 70 K / Temperature (min): 70 K
Image recordingElectron dose: 1.5 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameCategory
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.97 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 355832 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingB value: 68.91 / Protocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 6KPF

6kpf


Accession code: 6KPF / Source name: PDB / Type: experimental model

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