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- PDB-9m54: Cryo-EM structure of neuropeptide FF receptor 2 complex with NPVF -

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Basic information

Entry
Database: PDB / ID: 9m54
TitleCryo-EM structure of neuropeptide FF receptor 2 complex with NPVF
Components
  • (Guanine nucleotide-binding protein ...) x 3
  • (Neuropeptide ...) x 2
  • scfv16
KeywordsMEMBRANE PROTEIN/IMMUNE SYSTEM / GPCR / NPFFR2 / NPVF / NPFF / cryo-EM / MEMBRANE PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


opioid receptor binding / Orexin and neuropeptides FF and QRFP bind to their respective receptors / Adenylate cyclase inhibitory pathway / detection of abiotic stimulus / neuropeptide receptor activity / G-protein activation / Activation of the phototransduction cascade / Glucagon-type ligand receptors / Thromboxane signalling through TP receptor / Sensory perception of sweet, bitter, and umami (glutamate) taste ...opioid receptor binding / Orexin and neuropeptides FF and QRFP bind to their respective receptors / Adenylate cyclase inhibitory pathway / detection of abiotic stimulus / neuropeptide receptor activity / G-protein activation / Activation of the phototransduction cascade / Glucagon-type ligand receptors / Thromboxane signalling through TP receptor / Sensory perception of sweet, bitter, and umami (glutamate) taste / G beta:gamma signalling through PI3Kgamma / G beta:gamma signalling through CDC42 / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / Ca2+ pathway / G alpha (z) signalling events / High laminar flow shear stress activates signaling by PIEZO1 and PECAM1:CDH5:KDR in endothelial cells / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / Vasopressin regulates renal water homeostasis via Aquaporins / Adrenaline,noradrenaline inhibits insulin secretion / ADP signalling through P2Y purinoceptor 12 / G alpha (q) signalling events / G alpha (i) signalling events / Thrombin signalling through proteinase activated receptors (PARs) / Activation of G protein gated Potassium channels / G-protein activation / G beta:gamma signalling through PI3Kgamma / Prostacyclin signalling through prostacyclin receptor / G beta:gamma signalling through PLC beta / ADP signalling through P2Y purinoceptor 1 / Thromboxane signalling through TP receptor / Presynaptic function of Kainate receptors / G beta:gamma signalling through CDC42 / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / G alpha (12/13) signalling events / Glucagon-type ligand receptors / G beta:gamma signalling through BTK / ADP signalling through P2Y purinoceptor 12 / Adrenaline,noradrenaline inhibits insulin secretion / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / Ca2+ pathway / Thrombin signalling through proteinase activated receptors (PARs) / G alpha (z) signalling events / Extra-nuclear estrogen signaling / photoreceptor outer segment membrane / G alpha (s) signalling events / G alpha (q) signalling events / spectrin binding / G alpha (i) signalling events / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / High laminar flow shear stress activates signaling by PIEZO1 and PECAM1:CDH5:KDR in endothelial cells / Vasopressin regulates renal water homeostasis via Aquaporins / alkylglycerophosphoethanolamine phosphodiesterase activity / regulation of MAPK cascade / photoreceptor outer segment / neuropeptide signaling pathway / cellular response to hormone stimulus / positive regulation of protein localization to cell cortex / G protein-coupled serotonin receptor binding / cardiac muscle cell apoptotic process / photoreceptor inner segment / cellular response to forskolin / regulation of mitotic spindle organization / G protein-coupled receptor binding / G protein-coupled receptor activity / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / G-protein beta/gamma-subunit complex binding / GDP binding / cellular response to catecholamine stimulus / adenylate cyclase-activating dopamine receptor signaling pathway / cellular response to prostaglandin E stimulus / G-protein beta-subunit binding / heterotrimeric G-protein complex / sensory perception of taste / actin cytoskeleton / signaling receptor complex adaptor activity / retina development in camera-type eye / positive regulation of cytosolic calcium ion concentration / cell body / GTPase binding / midbody / cell cortex / phospholipase C-activating G protein-coupled receptor signaling pathway / G alpha (q) signalling events / cellular response to hypoxia / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / cell population proliferation / G protein-coupled receptor signaling pathway / cell division / GTPase activity / dendrite / synapse / centrosome / GTP binding / protein-containing complex binding / magnesium ion binding / nucleus / membrane / plasma membrane
Similarity search - Function
Neuropeptide FF receptor, type 2 / Neuropeptide FF receptor family / G-protein alpha subunit, group I / Serpentine type 7TM GPCR chemoreceptor Srsx / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G-alpha domain profile. / G protein alpha subunit / G-protein, gamma subunit ...Neuropeptide FF receptor, type 2 / Neuropeptide FF receptor family / G-protein alpha subunit, group I / Serpentine type 7TM GPCR chemoreceptor Srsx / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G-alpha domain profile. / G protein alpha subunit / G-protein, gamma subunit / G-protein gamma subunit domain profile. / G-protein gamma-like domain / G-protein gamma-like domain superfamily / GGL domain / G protein gamma subunit-like motifs / GGL domain / G protein beta WD-40 repeat protein / 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) / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Guanine nucleotide-binding protein G(i) subunit alpha-1 / Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / Neuropeptide FF receptor 2
Similarity search - Component
Biological speciesHomo sapiens (human)
Rattus norvegicus (Norway rat)
Bos taurus (domestic cattle)
Mus musculus (house mouse)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.24 Å
AuthorsPan, B.X. / Jiang, Y. / Li, X.Z.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: Cell Rep / Year: 2025
Title: Structural basis of peptide recognition and modulation for neuropeptide FF receptors.
Authors: Xinzhu Li / Heng Zhang / Wen Hu / Kai Wu / Shuai Li / Sanshan Jin / Yuling Yin / Qingning Yuan / H Eric Xu / Benxun Pan / Yi Jiang /
Abstract: Neuropeptide FF receptors 1 and 2 (NPFFR1 and NPFFR2) are RF-amide peptide receptors that couple to G proteins and regulate pain, opioid tolerance, and metabolism. Despite their physiological ...Neuropeptide FF receptors 1 and 2 (NPFFR1 and NPFFR2) are RF-amide peptide receptors that couple to G proteins and regulate pain, opioid tolerance, and metabolism. Despite their physiological significance, their ligand selectivity and activation mechanisms remain unclear. Using cryoelectron microscopy, we resolved four NPFFR1 and NPFFR2 structures bound to NPFF or NPVF, revealing conserved C-terminal RF-amide interactions within the orthosteric pocket and N-terminal variations driving subtype specificity. Structural and mutagenesis analyses identified ECL2 and the receptor N terminus as key determinants of NPVF-NPFFR1 and NPFF-NPFFR2 selectivity. Additionally, the structures elucidate the activation mechanism and uncover distinct G-coupling features between NPFFR subtypes. These findings provide molecular insights into peptide recognition and receptor activation within the RF-amide family, offering a structural framework for designing selective NPFFR modulators to treat pain, addiction, and metabolic disorders with enhanced specificity and reduced off-target effects.
History
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
R: Neuropeptide FF receptor 2
L: Neuropeptide VF
B: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
G: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
A: Guanine nucleotide-binding protein G(i) subunit alpha-1
S: scfv16


Theoretical massNumber of molelcules
Total (without water)163,2626
Polymers163,2626
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

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Neuropeptide ... , 2 types, 2 molecules RL

#1: Protein Neuropeptide FF receptor 2 / G-protein coupled receptor 74 / G-protein coupled receptor HLWAR77 / Neuropeptide G-protein coupled receptor


Mass: 48734.055 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: NPFFR2, GPR74, NPFF2, NPGPR / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9Y5X5
#2: Protein/peptide Neuropeptide VF


Mass: 969.140 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Guanine nucleotide-binding protein ... , 3 types, 3 molecules BGA

#3: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Transducin beta chain 1


Mass: 38975.578 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Gnb1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P54311
#4: 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.) Bos taurus (domestic cattle) / Gene: GNG2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P63212
#5: Protein Guanine nucleotide-binding protein G(i) subunit alpha-1 / Adenylate cyclase-inhibiting G alpha protein


Mass: 40445.059 Da / Num. of mol.: 1 / Mutation: G203A,A326S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bos taurus (domestic cattle) / Gene: GNAI1 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: P63097, Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement

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Antibody , 1 types, 1 molecules S

#6: Antibody scfv16


Mass: 26277.299 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Spodoptera frugiperda (fall armyworm)

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Details

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: NPFFR2 complex with heterotrimetic Gi-protein and neuropeptide NPVF
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Homo sapiens (human)9606
21Mus musculus (house mouse)10090
31Bos taurus (domestic cattle)9913
41Rattus norvegicus (Norway rat)10116
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.4
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: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 5000 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM software
IDNameCategoryImage processing-ID
1cryoSPARCparticle selection1
8PHENIXmodel refinement1
13cryoSPARC3D reconstruction1
14cryoSPARCparticle selection2
19cryoSPARC3D reconstruction2
Image processing
IDImage recording-ID
11
21
CTF correction
IDEM image processing-IDType
11PHASE FLIPPING AND AMPLITUDE CORRECTION
22PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.24 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 45511 / Symmetry type: POINT
Atomic model buildingSpace: REAL
RefinementHighest resolution: 3.24 Å

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