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- PDB-9ba0: Structural mechanism of CB1R binding to peripheral and biased inv... -

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Basic information

Entry
Database: PDB / ID: 9ba0
TitleStructural mechanism of CB1R binding to peripheral and biased inverse agonists
Components
  • CNb36
  • Cannabinoid receptor 1,Glycogen synthase
KeywordsSIGNALING PROTEIN/IMMUNE SYSTEM / obesity / membrane protein / nanobody / SIGNALING PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


cannabinoid signaling pathway / regulation of penile erection / negative regulation of dopamine secretion / retrograde trans-synaptic signaling by endocannabinoid / cannabinoid receptor activity / negative regulation of mast cell activation / alpha-1,4-glucan glucosyltransferase (UDP-glucose donor) activity / negative regulation of fatty acid beta-oxidation / negative regulation of serotonin secretion / positive regulation of acute inflammatory response to antigenic stimulus ...cannabinoid signaling pathway / regulation of penile erection / negative regulation of dopamine secretion / retrograde trans-synaptic signaling by endocannabinoid / cannabinoid receptor activity / negative regulation of mast cell activation / alpha-1,4-glucan glucosyltransferase (UDP-glucose donor) activity / negative regulation of fatty acid beta-oxidation / negative regulation of serotonin secretion / positive regulation of acute inflammatory response to antigenic stimulus / regulation of feeding behavior / regulation of presynaptic cytosolic calcium ion concentration / negative regulation of action potential / positive regulation of blood pressure / positive regulation of fever generation / Class A/1 (Rhodopsin-like receptors) / axonal fasciculation / regulation of metabolic process / regulation of synaptic transmission, GABAergic / G protein-coupled receptor signaling pathway, coupled to cyclic nucleotide second messenger / maternal process involved in female pregnancy / regulation of synaptic transmission, glutamatergic / negative regulation of blood pressure / response to nutrient / regulation of insulin secretion / GABA-ergic synapse / response to nicotine / response to cocaine / G protein-coupled receptor activity / positive regulation of neuron projection development / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / memory / adenylate cyclase-activating G protein-coupled receptor signaling pathway / glucose homeostasis / actin cytoskeleton / presynaptic membrane / growth cone / G alpha (i) signalling events / spermatogenesis / response to lipopolysaccharide / response to ethanol / mitochondrial outer membrane / positive regulation of apoptotic process / membrane raft / glutamatergic synapse / identical protein binding / plasma membrane / cytoplasm
Similarity search - Function
Cannabinoid receptor type 1 / Glycosyl transferases group 1 / Bacterial/plant glycogen synthase / Starch synthase, catalytic domain / Starch synthase catalytic domain / Cannabinoid receptor family / Serpentine type 7TM GPCR chemoreceptor Srsx / G-protein coupled receptors family 1 signature. / G protein-coupled receptor, rhodopsin-like / GPCR, rhodopsin-like, 7TM ...Cannabinoid receptor type 1 / Glycosyl transferases group 1 / Bacterial/plant glycogen synthase / Starch synthase, catalytic domain / Starch synthase catalytic domain / Cannabinoid receptor family / Serpentine type 7TM GPCR chemoreceptor Srsx / 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
: / Cannabinoid receptor 1 / Glycogen synthase
Similarity search - Component
Biological speciesHomo sapiens (human)
Pyrococcus abyssi GE5 (archaea)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.13 Å
AuthorsKumari, P. / Dvoracsko, S. / Enos, M.D. / Ramesh, K. / Lim, D. / Hassan, S.A. / Kunos, G. / Cinar, R. / Iyer, M.R. / Rosenbaum, D.M.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM116387 United States
CitationJournal: Nat Commun / Year: 2024
Title: Structural mechanism of CBR binding to peripheral and biased inverse agonists.
Authors: Punita Kumari / Szabolcs Dvorácskó / Michael D Enos / Karthik Ramesh / Darrix Lim / Sergio A Hassan / George Kunos / Resat Cinar / Malliga R Iyer / Daniel M Rosenbaum /
Abstract: The cannabinoid receptor 1 (CBR) regulates synaptic transmission in the central nervous system, but also has important roles in the peripheral organs controlling cellular metabolism. While earlier ...The cannabinoid receptor 1 (CBR) regulates synaptic transmission in the central nervous system, but also has important roles in the peripheral organs controlling cellular metabolism. While earlier generations of brain penetrant CBR antagonists advanced to the clinic for their effective treatment of obesity, such molecules were ultimately shown to exhibit negative effects on central reward pathways that thwarted their further therapeutic development. The peripherally restricted CBR inverse agonists MRI-1867 and MRI-1891 represent a new generation of compounds that retain the metabolic benefits of CBR inhibitors while sparing the negative psychiatric effects. To understand the mechanism of binding and inhibition of CBR by peripherally restricted antagonists, we developed a nanobody/fusion protein strategy for high-resolution cryo-EM structure determination of the GPCR inactive state, and used this method to determine structures of CBR bound to either MRI-1867 or MRI-1891. These structures reveal how these compounds retain high affinity and specificity for CBR's hydrophobic orthosteric site despite incorporating polar functionalities that lead to peripheral restriction. Further, the structure of the MRI-1891 complex along with accompanying molecular dynamics simulations shows how differential engagement with transmembrane helices and the proximal N-terminus can propagate through the receptor to contribute to biased inhibition of β-arrestin signaling.
History
DepositionApr 3, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 20, 2024Provider: repository / Type: Initial release
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Revision 1.1Nov 27, 2024Group: Data collection / Database references / Structure summary
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
R: Cannabinoid receptor 1,Glycogen synthase
N: CNb36
hetero molecules


Theoretical massNumber of molelcules
Total (without water)75,3813
Polymers74,7902
Non-polymers5911
Water00
1


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

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Components

#1: Protein Cannabinoid receptor 1,Glycogen synthase / CB-R / CB1 / CANN6


Mass: 60728.086 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Pyrococcus abyssi GE5 (archaea)
Gene: CNR1, CNR, PAB2292 / Strain: GE5 / Orsay / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P21554, UniProt: Q9V2J8
#2: Antibody CNb36


Mass: 14061.530 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)
#3: Chemical ChemComp-A1AKN / N-(N-{(E)-[(4S)-3-(4-chlorophenyl)-4-phenyl-4,5-dihydro-1H-pyrazol-1-yl][4-(trifluoromethyl)benzene-1-sulfonamido]methylidene}carbamimidoyl)acetamide


Mass: 591.004 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C26H22ClF3N6O3S
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: CB1-Nanobody complex / Type: COMPLEX / Entity ID: #1-#2 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMSodium ChlorideNaCl1
220 mMHEPES1
30.005 %Laurylmaltose neopentylglycol (LMNG)1
40.001 %CHS1
50.001 %Sodium Cholate1
SpecimenConc.: 2.75 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 1400 nm
Image recordingElectron dose: 1 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
2SerialEMimage acquisition
7PHENIXmodel fitting
9Cootmodel refinement
10RELION3.1initial Euler assignment
20PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1920000
3D reconstructionResolution: 3.13 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 99842 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0034847
ELECTRON MICROSCOPYf_angle_d0.4816565
ELECTRON MICROSCOPYf_dihedral_angle_d7.003709
ELECTRON MICROSCOPYf_chiral_restr0.039751
ELECTRON MICROSCOPYf_plane_restr0.004811

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