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- PDB-8tb7: Cryo-EM Structure of GPR61- -

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Basic information

Entry
Database: PDB / ID: 8tb7
TitleCryo-EM Structure of GPR61-
Components
  • Fab hinge-binding nanobody
  • Fab24 BAK5 heavy chain
  • Fab24 BAK5 light chain
  • G-protein coupled receptor 61,Soluble cytochrome b562
KeywordsSIGNALING PROTEIN / GPCR / inverse agonist / membrane protein / BRIL
Function / homology
Function and homology information


ligand-independent adenylate cyclase-activating G protein-coupled receptor signaling pathway / arrestin family protein binding / electron transport chain / G protein-coupled receptor activity / periplasmic space / electron transfer activity / receptor complex / endosome membrane / endosome / iron ion binding ...ligand-independent adenylate cyclase-activating G protein-coupled receptor signaling pathway / arrestin family protein binding / electron transport chain / G protein-coupled receptor activity / periplasmic space / electron transfer activity / receptor complex / endosome membrane / endosome / iron ion binding / G protein-coupled receptor signaling pathway / heme binding / plasma membrane
Similarity search - Function
Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / 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
Chem-ZOB / Soluble cytochrome b562 / G-protein coupled receptor 61
Similarity search - Component
Biological speciesHomo sapiens (human)
Escherichia coli (E. coli)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.94 Å
AuthorsLees, J.A. / Dias, J.M. / Han, S.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Commun / Year: 2023
Title: An inverse agonist of orphan receptor GPR61 acts by a G protein-competitive allosteric mechanism.
Authors: Joshua A Lees / João M Dias / Francis Rajamohan / Jean-Philippe Fortin / Rebecca O'Connor / Jimmy X Kong / Emily A G Hughes / Ethan L Fisher / Jamison B Tuttle / Gabrielle Lovett / Bethany ...Authors: Joshua A Lees / João M Dias / Francis Rajamohan / Jean-Philippe Fortin / Rebecca O'Connor / Jimmy X Kong / Emily A G Hughes / Ethan L Fisher / Jamison B Tuttle / Gabrielle Lovett / Bethany L Kormos / Rayomand J Unwalla / Lei Zhang / Anne-Marie Dechert Schmitt / Dahui Zhou / Michael Moran / Kimberly A Stevens / Kimberly F Fennell / Alison E Varghese / Andrew Maxwell / Emmaline E Cote / Yuan Zhang / Seungil Han /
Abstract: GPR61 is an orphan GPCR related to biogenic amine receptors. Its association with phenotypes relating to appetite makes it of interest as a druggable target to treat disorders of metabolism and body ...GPR61 is an orphan GPCR related to biogenic amine receptors. Its association with phenotypes relating to appetite makes it of interest as a druggable target to treat disorders of metabolism and body weight, such as obesity and cachexia. To date, the lack of structural information or a known biological ligand or tool compound has hindered comprehensive efforts to study GPR61 structure and function. Here, we report a structural characterization of GPR61, in both its active-like complex with heterotrimeric G protein and in its inactive state. Moreover, we report the discovery of a potent and selective small-molecule inverse agonist against GPR61 and structural elucidation of its allosteric binding site and mode of action. These findings offer mechanistic insights into an orphan GPCR while providing both a structural framework and tool compound to support further studies of GPR61 function and modulation.
History
DepositionJun 28, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 4, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
R: G-protein coupled receptor 61,Soluble cytochrome b562
H: Fab24 BAK5 heavy chain
N: Fab hinge-binding nanobody
L: Fab24 BAK5 light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)150,0795
Polymers149,5554
Non-polymers5251
Water181
1


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

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Components

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Protein , 1 types, 1 molecules R

#1: Protein G-protein coupled receptor 61,Soluble cytochrome b562


Mass: 59991.902 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Escherichia coli (E. coli)
Gene: GPR61, cybC / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9BZJ8, UniProt: P0ABE7

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Antibody , 3 types, 3 molecules HNL

#2: Antibody Fab24 BAK5 heavy chain


Mass: 52888.203 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
#3: Antibody Fab hinge-binding nanobody


Mass: 13276.541 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)
#4: Antibody Fab24 BAK5 light chain


Mass: 23398.066 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)

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Non-polymers , 2 types, 2 molecules

#5: Chemical ChemComp-ZOB / 6-{[(3,5-difluoropyridin-4-yl)methyl]amino}-N-(4-ethoxy-6-methylpyrimidin-2-yl)-2-methoxy-N-(2-methoxyethyl)pyridine-3-sulfonamide


Mass: 524.541 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C22H26F2N6O5S / Feature type: SUBJECT OF INVESTIGATION
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: H2O

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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: GPR61-ICL3-BRIL chimera in complex with anti-BRIL Fab24 BAK5 and hinge-binding nanobody bound to inverse agonist Compound 1
Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightExperimental value: NO
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
VitrificationCryogen name: ETHANE

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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 defocus max: 2400 nm / Nominal defocus min: 600 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.94 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 170451 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0036943
ELECTRON MICROSCOPYf_angle_d0.5979435
ELECTRON MICROSCOPYf_dihedral_angle_d4.774977
ELECTRON MICROSCOPYf_chiral_restr0.0481071
ELECTRON MICROSCOPYf_plane_restr0.0041209

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