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- PDB-9jg0: Cryo-EM structure of neuropeptide FF receptor 2 in the ligand-fre... -

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Basic information

Entry
Database: PDB / ID: 9jg0
TitleCryo-EM structure of neuropeptide FF receptor 2 in the ligand-free state with BRIL fusion, anti-BRIL Fab, and nanobody
Components
  • Anti-BRIL fab heavy chain
  • Anti-BRIL fab light chain
  • Anti-fab nanobody
  • Isoform 2 of Neuropeptide FF receptor 2,Soluble cytochrome b562
KeywordsMEMBRANE PROTEIN / GPCR
Function / homology
Function and homology information


opioid receptor binding / Orexin and neuropeptides FF and QRFP bind to their respective receptors / detection of abiotic stimulus / neuropeptide receptor activity / regulation of MAPK cascade / neuropeptide signaling pathway / cellular response to hormone stimulus / electron transport chain / G protein-coupled receptor activity / actin cytoskeleton ...opioid receptor binding / Orexin and neuropeptides FF and QRFP bind to their respective receptors / detection of abiotic stimulus / neuropeptide receptor activity / regulation of MAPK cascade / neuropeptide signaling pathway / cellular response to hormone stimulus / electron transport chain / G protein-coupled receptor activity / actin cytoskeleton / G alpha (q) signalling events / electron transfer activity / periplasmic space / G protein-coupled receptor signaling pathway / iron ion binding / heme binding / plasma membrane
Similarity search - Function
Neuropeptide FF receptor family / Neuropeptide FF receptor, type 2 / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / 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
Soluble cytochrome b562 / Neuropeptide FF receptor 2
Similarity search - Component
Biological speciesHomo sapiens (human)
Escherichia coli (E. coli)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.91 Å
AuthorsKim, J. / Choi, H.-J.
Funding support Korea, Republic Of, 1items
OrganizationGrant numberCountry
National Research Foundation (NRF, Korea) Korea, Republic Of
CitationJournal: EMBO Rep / Year: 2025
Title: Structural insights into the selective recognition of RF-amide peptides by neuropeptide FF receptor 2.
Authors: Jeesoo Kim / Sooyoung Hong / Hajin Lee / Hyun Sik Lee / Chaehee Park / Jinuk Kim / Wonpil Im / Hee-Jung Choi /
Abstract: Neuropeptide FF Receptor 2 (NPFFR2), a G-protein-coupled receptor, plays a role in pain modulation and diet-induced thermogenesis. While NPFFR2 is strongly activated by neuropeptides FF (NPFFs), it ...Neuropeptide FF Receptor 2 (NPFFR2), a G-protein-coupled receptor, plays a role in pain modulation and diet-induced thermogenesis. While NPFFR2 is strongly activated by neuropeptides FF (NPFFs), it shows low activity in response to RF-amide-related peptides (RFRPs), despite the peptides belonging to a shared family. In contrast, NPFFR1, which shares high sequence similarity with NPFFR2, is activated by RFRPs and regulates reproductive hormone balance. The molecular basis for these receptor-specific interactions with their RF-amide peptides remains unclear. Here, we present cryo-electron microscopy structures of NPFFR2 in its active state bound to the agonist RF-amide peptide hNPSF, and in its ligand-free state. Structural analysis reveals that the C-terminal RF-amide moiety engages conserved residues in the transmembrane domain, while the N-terminal segment interacts in a receptor subtype-specific manner. Key selectivity-determining residues in NPFFR2 are also identified. A homology model of NPFFR1 bound to RFRP, supported by mutagenesis studies, further validates this selectivity mechanism. Additionally, structural comparison between the inactive and active states of NPFFR2 suggests a TM3-mediated activation mechanism. These findings provide insights into RF-amide peptide recognition by NPFF receptors.
History
DepositionSep 5, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 9, 2025Provider: repository / Type: Initial release
Revision 1.0Apr 9, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Apr 9, 2025Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Apr 9, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Apr 9, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 9, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 9, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Apr 9, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
R: Isoform 2 of Neuropeptide FF receptor 2,Soluble cytochrome b562
H: Anti-BRIL fab heavy chain
K: Anti-fab nanobody
L: Anti-BRIL fab light chain


Theoretical massNumber of molelcules
Total (without water)122,7374
Polymers122,7374
Non-polymers00
Water00
1


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

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Components

#1: Protein Isoform 2 of Neuropeptide FF receptor 2,Soluble cytochrome b562 / G-protein coupled receptor 74 / G-protein coupled receptor HLWAR77 / Neuropeptide G-protein coupled ...G-protein coupled receptor 74 / G-protein coupled receptor HLWAR77 / Neuropeptide G-protein coupled receptor / Cytochrome b-562


Mass: 60471.199 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: 246 to 363 is soluble cytochrome b562 with linker
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Escherichia coli (E. coli)
Gene: NPFFR2, GPR74, NPFF2, NPGPR, cybC / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9Y5X5, UniProt: P0ABE7
#2: Antibody Anti-BRIL fab heavy chain


Mass: 24321.084 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Trichoplusia ni (cabbage looper)
#3: Antibody Anti-fab nanobody


Mass: 14461.796 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#4: Antibody Anti-BRIL fab light chain


Mass: 23483.062 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Trichoplusia ni (cabbage looper)
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: human neuropeptide FF receptor 2 in the apo state with BRIL fusion, anti-BRIL Fab, and nanobody
Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightValue: 0.12 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 8
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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.91 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 324660 / Symmetry type: POINT

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