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- PDB-6kpg: Cryo-EM structure of CB1-G protein complex -

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Basic information

Entry
Database: PDB / ID: 6kpg
TitleCryo-EM structure of CB1-G protein complex
Components
  • (Guanine nucleotide-binding protein ...) x 3
  • Cannabinoid receptor 1Cannabinoid receptor type 1
  • scFv16
KeywordsMEMBRANE PROTEIN / GPCR / G protein / cryo-EM
Function / homology
Function and homology information


cannabinoid signaling pathway / regulation of penile erection / retrograde trans-synaptic signaling by endocannabinoid / cannabinoid receptor activity / negative regulation of mast cell activation / trans-synaptic signaling by endocannabinoid, modulating synaptic transmission / negative regulation of fatty acid beta-oxidation / negative regulation of dopamine secretion / positive regulation of acute inflammatory response to antigenic stimulus / regulation of presynaptic cytosolic calcium ion concentration ...cannabinoid signaling pathway / regulation of penile erection / retrograde trans-synaptic signaling by endocannabinoid / cannabinoid receptor activity / negative regulation of mast cell activation / trans-synaptic signaling by endocannabinoid, modulating synaptic transmission / negative regulation of fatty acid beta-oxidation / negative regulation of dopamine secretion / positive regulation of acute inflammatory response to antigenic stimulus / regulation of presynaptic cytosolic calcium ion concentration / regulation of feeding behavior / negative regulation of serotonin secretion / negative regulation of action potential / Class A/1 (Rhodopsin-like receptors) / positive regulation of blood pressure / positive regulation of fever generation / regulation of metabolic process / axonal fasciculation / regulation of synaptic transmission, GABAergic / G protein-coupled receptor signaling pathway, coupled to cyclic nucleotide second messenger / regulation of insulin secretion / Adenylate cyclase inhibitory pathway / GABA-ergic synapse / positive regulation of protein localization to cell cortex / maternal process involved in female pregnancy / regulation of cAMP-mediated signaling / D2 dopamine receptor binding / G protein-coupled serotonin receptor binding / regulation of mitotic spindle organization / cellular response to forskolin / regulation of synaptic transmission, glutamatergic / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / negative regulation of blood pressure / response to nutrient / response to cocaine / response to nicotine / Regulation of insulin secretion / G protein-coupled receptor binding / G protein-coupled receptor activity / 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 / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / G-protein activation / G protein-coupled acetylcholine receptor signaling pathway / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / Prostacyclin signalling through prostacyclin receptor / Glucagon signaling in metabolic regulation / G beta:gamma signalling through CDC42 / adenylate cyclase-activating G protein-coupled receptor signaling pathway / positive regulation of neuron projection development / ADP signalling through P2Y purinoceptor 12 / G beta:gamma signalling through BTK / Sensory perception of sweet, bitter, and umami (glutamate) taste / memory / response to peptide hormone / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / photoreceptor disc membrane / Adrenaline,noradrenaline inhibits insulin secretion / Glucagon-type ligand receptors / Vasopressin regulates renal water homeostasis via Aquaporins / G alpha (z) signalling events / cellular response to catecholamine stimulus / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / ADORA2B mediated anti-inflammatory cytokines production / adenylate cyclase-activating dopamine receptor signaling pathway / ADP signalling through P2Y purinoceptor 1 / G beta:gamma signalling through PI3Kgamma / cellular response to prostaglandin E stimulus / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / sensory perception of taste / GPER1 signaling / GDP binding / G-protein beta-subunit binding / heterotrimeric G-protein complex / Inactivation, recovery and regulation of the phototransduction cascade / extracellular vesicle / G alpha (12/13) signalling events / signaling receptor complex adaptor activity / actin cytoskeleton / Thrombin signalling through proteinase activated receptors (PARs) / glucose homeostasis / retina development in camera-type eye / GTPase binding / presynaptic membrane / Ca2+ pathway / phospholipase C-activating G protein-coupled receptor signaling pathway / cell cortex / midbody / G alpha (i) signalling events / growth cone / fibroblast proliferation / G alpha (s) signalling events / G alpha (q) signalling events / spermatogenesis / response to ethanol
Similarity search - Function
Cannabinoid receptor type 1 / Cannabinoid receptor family / Transducin (heterotrimeric G protein), gamma chain / G Protein Gi Gamma 2 / YVTN repeat-like/Quinoprotein amine dehydrogenase / G-protein alpha subunit, group I / 7 Propeller / Methylamine Dehydrogenase; Chain H / Serpentine type 7TM GPCR chemoreceptor Srsx / G-alpha domain profile. ...Cannabinoid receptor type 1 / Cannabinoid receptor family / Transducin (heterotrimeric G protein), gamma chain / G Protein Gi Gamma 2 / YVTN repeat-like/Quinoprotein amine dehydrogenase / G-protein alpha subunit, group I / 7 Propeller / Methylamine Dehydrogenase; Chain H / Serpentine type 7TM GPCR chemoreceptor Srsx / G-alpha domain profile. / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G protein alpha subunit / G-protein, gamma subunit / G-protein gamma subunit domain profile. / GGL domain / G-protein gamma-like domain superfamily / G-protein gamma-like domain / GGL domain / G protein gamma subunit-like motifs / 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) / Few Secondary Structures / Irregular / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / Immunoglobulins / Immunoglobulin-like / Sandwich / P-loop containing nucleoside triphosphate hydrolase / Mainly Beta
Similarity search - Domain/homology
Chem-8D0 / Cannabinoid receptor 1 / 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: 3 Å
AuthorsHua, T. / Li, X.T. / Wu, L.J. / Makriyannis, A. / Wang, Y.X. / Shen, L. / Liu, Z.J.
CitationJournal: Cell / Year: 2020
Title: Activation and Signaling Mechanism Revealed by Cannabinoid Receptor-G Complex Structures.
Authors: Tian Hua / Xiaoting Li / Lijie Wu / Christos Iliopoulos-Tsoutsouvas / Yuxia Wang / Meng Wu / Ling Shen / Christina A Brust / Spyros P Nikas / Feng Song / Xiyong Song / Shuguang Yuan / ...Authors: Tian Hua / Xiaoting Li / Lijie Wu / Christos Iliopoulos-Tsoutsouvas / Yuxia Wang / Meng Wu / Ling Shen / Christina A Brust / Spyros P Nikas / Feng Song / Xiyong Song / Shuguang Yuan / Qianqian Sun / Yiran Wu / Shan Jiang / Travis W Grim / Othman Benchama / Edward L Stahl / Nikolai Zvonok / Suwen Zhao / Laura M Bohn / Alexandros Makriyannis / Zhi-Jie Liu /
Abstract: Human endocannabinoid systems modulate multiple physiological processes mainly through the activation of cannabinoid receptors CB1 and CB2. Their high sequence similarity, low agonist selectivity, ...Human endocannabinoid systems modulate multiple physiological processes mainly through the activation of cannabinoid receptors CB1 and CB2. Their high sequence similarity, low agonist selectivity, and lack of activation and G protein-coupling knowledge have hindered the development of therapeutic applications. Importantly, missing structural information has significantly held back the development of promising CB2-selective agonist drugs for treating inflammatory and neuropathic pain without the psychoactivity of CB1. Here, we report the cryoelectron microscopy structures of synthetic cannabinoid-bound CB2 and CB1 in complex with G, as well as agonist-bound CB2 crystal structure. Of important scientific and therapeutic benefit, our results reveal a diverse activation and signaling mechanism, the structural basis of CB2-selective agonists design, and the unexpected interaction of cholesterol with CB1, suggestive of its endogenous allosteric modulating role.
History
DepositionAug 15, 2019Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Feb 12, 2020Provider: repository / Type: Initial release
Revision 1.1Mar 11, 2020Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first

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Structure visualization

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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 1
S: scFv16
hetero molecules


Theoretical massNumber of molelcules
Total (without water)167,2236
Polymers166,7775
Non-polymers4461
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area11170 Å2
ΔGint-71 kcal/mol
Surface area46840 Å2

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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: 40283.836 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


Mass: 38045.629 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


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 1 / Cannabinoid receptor type 1 / CB1 / CANN6


Mass: 52843.691 Da / Num. of mol.: 1 / Mutation: E273K, T283V, R340E
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CNR1, CNR
Production host: Mammalian expression vector HA-MCS-pcDNA3.1 (others)
References: UniProt: P21554
#5: Antibody scFv16


Mass: 27742.861 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-8D0 / (6~{a}~{R},9~{R},10~{a}~{R})-9-(hydroxymethyl)-3-(8-isothiocyanato-2-methyl-octan-2-yl)-6,6-dimethyl-6~{a},7,8,9,10,10~{a}-hexahydrobenzo[c]chromen-1-ol


Mass: 445.658 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C26H39NO3S / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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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: CB1-G protein complex / 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
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 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 FIELDBright-field microscopy / Nominal magnification: 130000 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: 1.333 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 5577
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansWidth: 7420 / Height: 7676 / Movie frames/image: 45 / Used frames/image: 1-45

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEM3.7.3image acquisition
4Gctf1.06CTF correction
7UCSF Chimeramodel fitting
9RELION3.03initial Euler assignment
10RELION3.03final Euler assignment
11RELION3.03classification
12RELION3.033D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2186340
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 505644 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingB value: 72.4 / Protocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 6N4B

6n4b

Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0138988
ELECTRON MICROSCOPYf_angle_d1.04912177
ELECTRON MICROSCOPYf_dihedral_angle_d11.4755319
ELECTRON MICROSCOPYf_chiral_restr0.0631399
ELECTRON MICROSCOPYf_plane_restr0.0071524

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