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- EMDB-61947: Cryo-EM structure of UTP-bound P2Y purinoceptor 4-miniGq-Nb35 complex -

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Entry
Database: EMDB / ID: EMD-61947
TitleCryo-EM structure of UTP-bound P2Y purinoceptor 4-miniGq-Nb35 complex
Map data
Sample
  • Complex: Cryo-EM structure of UTP-bound P2Y purinoceptor 4-miniGq-Nb35 complex
    • Protein or peptide: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
    • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
    • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
    • Protein or peptide: Nanobody 35
    • Protein or peptide: P2Y purinoceptor 4
  • Ligand: URIDINE 5'-TRIPHOSPHATE
KeywordsG protein-coupled receptors / G-protein signaling / Nucleotide receptors / MEMBRANE PROTEIN
Function / homology
Function and homology information


G protein-coupled UTP receptor activity / P2Y receptors / G protein-coupled purinergic nucleotide receptor activity / transepithelial chloride transport / regulation of presynaptic cytosolic calcium ion concentration / cellular response to ATP / regulation of synaptic vesicle exocytosis / PKA activation in glucagon signalling / hair follicle placode formation / developmental growth ...G protein-coupled UTP receptor activity / P2Y receptors / G protein-coupled purinergic nucleotide receptor activity / transepithelial chloride transport / regulation of presynaptic cytosolic calcium ion concentration / cellular response to ATP / regulation of synaptic vesicle exocytosis / PKA activation in glucagon signalling / hair follicle placode formation / developmental growth / D1 dopamine receptor binding / intracellular transport / vascular endothelial cell response to laminar fluid shear stress / renal water homeostasis / Hedgehog 'off' state / adenylate cyclase-activating adrenergic receptor signaling pathway / activation of adenylate cyclase activity / presynaptic active zone membrane / regulation of insulin secretion / cellular response to glucagon stimulus / adenylate cyclase activator activity / trans-Golgi network membrane / negative regulation of inflammatory response to antigenic stimulus / bone development / G-protein beta/gamma-subunit complex binding / platelet aggregation / Olfactory Signaling Pathway / cognition / Activation of the phototransduction cascade / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / G protein-coupled acetylcholine receptor signaling pathway / adenylate cyclase-activating G protein-coupled receptor signaling pathway / G-protein activation / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / Prostacyclin signalling through prostacyclin receptor / G beta:gamma signalling through CDC42 / Glucagon signaling in metabolic regulation / G beta:gamma signalling through BTK / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / ADP signalling through P2Y purinoceptor 12 / Sensory perception of sweet, bitter, and umami (glutamate) taste / photoreceptor disc membrane / Glucagon-type ligand receptors / Adrenaline,noradrenaline inhibits insulin secretion / Vasopressin regulates renal water homeostasis via Aquaporins / G alpha (z) signalling events / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / cellular response to catecholamine stimulus / ADORA2B mediated anti-inflammatory cytokines production / ADP signalling through P2Y purinoceptor 1 / G beta:gamma signalling through PI3Kgamma / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / adenylate cyclase-activating dopamine receptor signaling pathway / sensory perception of smell / GPER1 signaling / Inactivation, recovery and regulation of the phototransduction cascade / cellular response to prostaglandin E stimulus / G-protein beta-subunit binding / heterotrimeric G-protein complex / G alpha (12/13) signalling events / sensory perception of taste / extracellular vesicle / signaling receptor complex adaptor activity / Thrombin signalling through proteinase activated receptors (PARs) / positive regulation of cold-induced thermogenesis / G protein activity / GTPase binding / positive regulation of cytosolic calcium ion concentration / Ca2+ pathway / retina development in camera-type eye / High laminar flow shear stress activates signaling by PIEZO1 and PECAM1:CDH5:KDR in endothelial cells / fibroblast proliferation / G alpha (i) signalling events / basolateral plasma membrane / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / G alpha (s) signalling events / phospholipase C-activating G protein-coupled receptor signaling pathway / G alpha (q) signalling events / Ras protein signal transduction / Extra-nuclear estrogen signaling / cell population proliferation / G protein-coupled receptor signaling pathway / apical plasma membrane / lysosomal membrane / GTPase activity / synapse / protein-containing complex binding / GTP binding / glutamatergic synapse / signal transduction / extracellular exosome / ATP binding / metal ion binding / membrane / plasma membrane / cytosol / cytoplasm
Similarity search - Function
P2Y purinoceptor 4 / G-protein alpha subunit, group S / 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 ...P2Y purinoceptor 4 / G-protein alpha subunit, group S / 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 / 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. / WD domain, G-beta repeat / 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
P2Y purinoceptor 4 / Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
Similarity search - Component
Biological speciesHomo sapiens (human) / Vicugna pacos (alpaca)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.14 Å
AuthorsLan B / Zhang S / Liu X / Lin B
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: Cell Discov / Year: 2025
Title: Structural insight into the self-activation and G-protein coupling of P2Y2 receptor.
Authors: Baoliang Lan / Shuhao Zhang / Kai Chen / Shengjie Dai / Jiaqi Fei / Kaixuan Gao / Xiaoou Sun / Bin Lin / Xiangyu Liu /
Abstract: Purinergic P2Y2 receptor (P2Y2R) represents a typically extracellular ATP and UTP sensor for mediating purinergic signaling. Despite its importance as a pharmacological target, the molecular ...Purinergic P2Y2 receptor (P2Y2R) represents a typically extracellular ATP and UTP sensor for mediating purinergic signaling. Despite its importance as a pharmacological target, the molecular mechanisms underlying ligand recognition and G-protein coupling have remained elusive due to lack of structural information. In this study, we determined the cryo-electron microscopy (cryo-EM) structures of the apo P2Y2R in complex with G, ATP-bound P2Y2R in complex with G or G, and UTP-bound P2Y4R in complex with G. These structures reveal the similarities and distinctions of ligand recognition within the P2Y receptor family. Furthermore, a comprehensive analysis of G-protein coupling reveals that P2Y2R exhibits promiscuity in coupling with both G and G proteins. Combining molecular dynamics simulations and signaling assays, we elucidate the molecular mechanisms by which P2Y2R differentiates pathway-specific G or G coupling through distinct structural components on the intracellular side. Strikingly, we identify a helix-like segment within the N-terminus that occupies the orthosteric ligand-binding pocket of P2Y2R, accounting for its self-activation. Taken together, these findings provide a molecular framework for understanding the activation mechanism of P2Y2R, encompassing ligand recognition, G-protein coupling, and a novel N-terminus-mediated self-activation mechanism.
History
DepositionOct 15, 2024-
Header (metadata) releaseJun 11, 2025-
Map releaseJun 11, 2025-
UpdateJun 11, 2025-
Current statusJun 11, 2025Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_61947.png

Downloads & links

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Map

FileDownload / File: emd_61947.map.gz / Format: CCP4 / Size: 45.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 228 pix.
= 246.81 Å		1.08 Å/pix.
x 228 pix.
= 246.81 Å		1.08 Å/pix.
x 228 pix.
= 246.81 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.0825 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.51
Minimum - Maximum-6.031717 - 8.125833500000001
Average (Standard dev.)-0.0021177053 (±0.1310321)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions228228228
Spacing228228228
CellA=B=C: 246.81 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_61947_half_map_1.map
Projections & Slices
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Half map: #1

Fileemd_61947_half_map_2.map
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Sample components

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Entire : Cryo-EM structure of UTP-bound P2Y purinoceptor 4-miniGq-Nb35 complex

EntireName: Cryo-EM structure of UTP-bound P2Y purinoceptor 4-miniGq-Nb35 complex
Components
  • Complex: Cryo-EM structure of UTP-bound P2Y purinoceptor 4-miniGq-Nb35 complex
    • Protein or peptide: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
    • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
    • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
    • Protein or peptide: Nanobody 35
    • Protein or peptide: P2Y purinoceptor 4
  • Ligand: URIDINE 5'-TRIPHOSPHATE

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Supramolecule #1: Cryo-EM structure of UTP-bound P2Y purinoceptor 4-miniGq-Nb35 complex

SupramoleculeName: Cryo-EM structure of UTP-bound P2Y purinoceptor 4-miniGq-Nb35 complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short

MacromoleculeName: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
type: protein_or_peptide / ID: 1
Details: The N-terminal sequence of Gi1 is incorporated into the mini-G protein;Certain residues mutated to match Guanine nucleotide-binding protein G(q) subunit
Number of copies: 1 / Enantiomer: LEVO
EC number: Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 28.01476 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: TLSAEDKAAV ERSKMIEKQL QKDKQVYRAT HRLLLLGADN SGKSTIVKQM RILHGGSGGS GGTSGIFETK FQVDKVNFHM FDVGGQRDE RRKWIQCFND VTAIIFVVDS SDYNRLQEAL NDFKSIWNNR WLRTISVILF LNKQDLLAEK VLAGKSKIED Y FPEFARYT ...String:
TLSAEDKAAV ERSKMIEKQL QKDKQVYRAT HRLLLLGADN SGKSTIVKQM RILHGGSGGS GGTSGIFETK FQVDKVNFHM FDVGGQRDE RRKWIQCFND VTAIIFVVDS SDYNRLQEAL NDFKSIWNNR WLRTISVILF LNKQDLLAEK VLAGKSKIED Y FPEFARYT TPEDATPEPG EDPRVTRAKY FIRDEFLRIS TASGDGRHYC YPHFTCAVDT ENARRIFNDC KDIILQMNLR EY NLV

UniProtKB: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short, Guanine nucleotide-binding protein G(s) subunit alpha isoforms short

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Macromolecule #2: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1

MacromoleculeName: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 39.418086 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MHHHHHHLEV LFQGPGSSGS ELDQLRQEAE QLKNQIRDAR KACADATLSQ ITNNIDPVGR IQMRTRRTLR GHLAKIYAMH WGTDSRLLV SASQDGKLII WDSYTTNKVH AIPLRSSWVM TCAYAPSGNY VACGGLDNIC SIYNLKTREG NVRVSRELAG H TGYLSCCR ...String:
MHHHHHHLEV LFQGPGSSGS ELDQLRQEAE QLKNQIRDAR KACADATLSQ ITNNIDPVGR IQMRTRRTLR GHLAKIYAMH WGTDSRLLV SASQDGKLII WDSYTTNKVH AIPLRSSWVM TCAYAPSGNY VACGGLDNIC SIYNLKTREG NVRVSRELAG H TGYLSCCR FLDDNQIVTS SGDTTCALWD IETGQQTTTF TGHTGDVMSL SLAPDTRLFV SGACDASAKL WDVREGMCRQ TF TGHESDI NAICFFPNGN AFATGSDDAT CRLFDLRADQ ELMTYSHDNI ICGITSVSFS KSGRLLLAGY DDFNCNVWDA LKA DRAGVL AGHDNRVSCL GVTDDGMAVA TGSWDSFLKI WN

UniProtKB: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1

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Macromolecule #3: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2

MacromoleculeName: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 7.861143 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString:
MASNNTASIA QARKLVEQLK MEANIDRIKV SKAAADLMAY CEAHAKEDPL LTPVPASENP FREKKFFCAI L

UniProtKB: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2

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Macromolecule #4: Nanobody 35

MacromoleculeName: Nanobody 35 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Vicugna pacos (alpaca)
Molecular weightTheoretical: 14.686328 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
QVQLQESGGG LVQPGGSLRL SCAASGFTFS NYKMNWVRQA PGKGLEWVSD ISQSGASISY TGSVKGRFTI SRDNAKNTLY LQMNSLKPE DTAVYYCARC PAPFTPFCFD VTSTTYAYRG QGTQVTVSSH HHHHH

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Macromolecule #5: P2Y purinoceptor 4

MacromoleculeName: P2Y purinoceptor 4 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 45.787227 KDa
Recombinant expressionOrganism: Homo (humans)
SequenceString: MKTIIALSYI FCLVFADYKD DDDAMASTES SLLRSLGLSP GPGSSEVELD CWFDEDFKFI LLPVSYAVVF VLGLGLNAPT LWLFIFRLR PWDATATYMF HLALSDTLYV LSLPTLIYYY AAHNHWPFGT EICKFVRFLF YWNLYCSVLF LTCISVHRYL G ICHPLRAL ...String:
MKTIIALSYI FCLVFADYKD DDDAMASTES SLLRSLGLSP GPGSSEVELD CWFDEDFKFI LLPVSYAVVF VLGLGLNAPT LWLFIFRLR PWDATATYMF HLALSDTLYV LSLPTLIYYY AAHNHWPFGT EICKFVRFLF YWNLYCSVLF LTCISVHRYL G ICHPLRAL RWGRPRLAGL LCLAVWLVVA GCLVPNLFFV TTSNKGTTVL CHDTTRPEEF DHYVHFSSAV MGLLFGVPCL VT LVCYGLM ARRLYQPLPG SAQSSSRLRS LRTIAVVLTV FAVCFVPFHI TRTIYYLARL LEADCRVLNI VNVVYKVTRP LAS ANSCLD PVLYLLTGDK YRRQLRQLCG GGKPQPRTAA SSLALVSLPE DSSCRWAATP QDSSCSTPRA DRLHHHHHHG GSGG LEVLF QGP

UniProtKB: P2Y purinoceptor 4

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Macromolecule #6: URIDINE 5'-TRIPHOSPHATE

MacromoleculeName: URIDINE 5'-TRIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 1 / Formula: UTP
Molecular weightTheoretical: 484.141 Da
Chemical component information

ChemComp-UTP:
URIDINE 5'-TRIPHOSPHATE / UTP*YM

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.6 µm / Nominal defocus min: 1.2 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.14 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 356473
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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