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- EMDB-44394: Structural mechanism of CB1R binding to peripheral and biased inv... -

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Basic information

Entry
Database: EMDB / ID: EMD-44394
TitleStructural mechanism of CB1R binding to peripheral and biased inverse agonists
Map data
Sample
  • Complex: CB1-Nanobody complex
    • Protein or peptide: Cannabinoid receptor 1,Glycogen synthase
    • Protein or peptide: CNb36
  • Ligand: N-(N-{(E)-[(4S)-3-(4-chlorophenyl)-4-phenyl-4,5-dihydro-1H-pyrazol-1-yl][4-(trifluoromethyl)benzene-1-sulfonamido]methylidene}carbamimidoyl)acetamide
Keywordsobesity / 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) / Lama glama (llama)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.13 Å
AuthorsKumari P / Dvoracsko S / Enos MD / Ramesh K / Lim D / Hassan SA / Kunos G / Cinar R / Iyer MR / Rosenbaum DM
Funding support United States, 1 items
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, 2024-
Header (metadata) releaseNov 20, 2024-
Map releaseNov 20, 2024-
UpdateMay 28, 2025-
Current statusMay 28, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44394.png

Downloads & links

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Map

FileDownload / File: emd_44394.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 200 pix.
= 214. Å		1.07 Å/pix.
x 200 pix.
= 214. Å		1.07 Å/pix.
x 200 pix.
= 214. Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0384
Minimum - Maximum-0.10851012 - 0.18998335
Average (Standard dev.)0.000056224195 (±0.0049568964)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 214.00002 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_44394_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_44394_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : CB1-Nanobody complex

EntireName: CB1-Nanobody complex
Components
  • Complex: CB1-Nanobody complex
    • Protein or peptide: Cannabinoid receptor 1,Glycogen synthase
    • Protein or peptide: CNb36
  • Ligand: N-(N-{(E)-[(4S)-3-(4-chlorophenyl)-4-phenyl-4,5-dihydro-1H-pyrazol-1-yl][4-(trifluoromethyl)benzene-1-sulfonamido]methylidene}carbamimidoyl)acetamide

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Supramolecule #1: CB1-Nanobody complex

SupramoleculeName: CB1-Nanobody complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Cannabinoid receptor 1,Glycogen synthase

MacromoleculeName: Cannabinoid receptor 1,Glycogen synthase / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 60.728086 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: DYKDDDDAMD QVNITEFYNK SLSSFENLYF QGGIQCGENF MDIECFMVLN PSQQLAIAVL SLTLGTFTVL ENLLVLCVIL HSRSLRCRP SYHFIGSLAV ADLLGSVIFV YSFIDFHVFH RKDSRNVFLF KLGGVTASFT AKVGSLFLAA IDRYISIHRP L AYKRIVTR ...String:
DYKDDDDAMD QVNITEFYNK SLSSFENLYF QGGIQCGENF MDIECFMVLN PSQQLAIAVL SLTLGTFTVL ENLLVLCVIL HSRSLRCRP SYHFIGSLAV ADLLGSVIFV YSFIDFHVFH RKDSRNVFLF KLGGVTASFT AKVGSLFLAA IDRYISIHRP L AYKRIVTR PKAVVAFCLM WTIAIVIAVL PLLGWNCEKL QSVCSDIFPH IDKTYLMFWI GVVSVLLLFI VYAYMYILWK AG IDCSFWN ESYLTGSRDE RKKSLLSKFG MDEGVTFMFI GRFDRGQKGV DVLLKAIEIL SSKKEFQEMR FIIIGKGDPE LEG WARSLE EKHGNVKVIT EMLSREFVRE LYGSVDFVII PSYFEPFGLV ALEAMCLGAI PIASAVGGLR DIITNETGIL VKAG DPGEL ANAILKALEL SRSDLSKFRE NCKKRAMSFS DQARMDIELA KTLVLILVVL IICWGPLLAI MVYDVFGKMN KLIKT VFAF CSMLCLLNST VNPIIYALRS KDLRHAFRSM FPSCENLYFQ GHHHHHHHHH H

UniProtKB: Cannabinoid receptor 1, Glycogen synthase, Cannabinoid receptor 1

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Macromolecule #2: CNb36

MacromoleculeName: CNb36 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 14.06153 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
QVQLQESGGG LVQAGGSLRL SCAASGTIFG PDVMGWYRQA PGKERELVAG ISNGANTYYA DSVKGRFTIS RDNAKNTVYL QMNSLKPED TAVYYCAAEV LDYTFAYLYH AYWGQGTQVT VSSHHHHHH

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Macromolecule #3: N-(N-{(E)-[(4S)-3-(4-chlorophenyl)-4-phenyl-4,5-dihydro-1H-pyrazo...

MacromoleculeName: N-(N-{(E)-[(4S)-3-(4-chlorophenyl)-4-phenyl-4,5-dihydro-1H-pyrazol-1-yl][4-(trifluoromethyl)benzene-1-sulfonamido]methylidene}carbamimidoyl)acetamide
type: ligand / ID: 3 / Number of copies: 1 / Formula: A1AKN
Molecular weightTheoretical: 591.004 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration2.75 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
150.0 mMNaClSodium Chloride
20.0 mMHEPES
0.005 %Laurylmaltose neopentylglycol (LMNG)
0.001 %CHS
0.001 %Sodium Cholate
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 1.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.4000000000000001 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1920000
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.13 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 99842
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: PROJECTION MATCHING
FSC plot (resolution estimation)

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