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- PDB-8z9o: Cryo-EM structure of human GPR4-Gs complex -

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Basic information

Entry
Database: PDB / ID: 8z9o
TitleCryo-EM structure of human GPR4-Gs complex
Components
  • (Guanine nucleotide-binding protein ...) x 2
  • G-protein coupled receptor 4
  • Isoform Gnas-2 of Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
  • Nanobody-35
KeywordsMEMBRANE PROTEIN/IMMUNE SYSTEM / GPCR / class A / GPR4-Gs / cryo-EM / protein sensing / active state / MEMBRANE PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


regulation of vascular permeability / Class A/1 (Rhodopsin-like receptors) / 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 ...regulation of vascular permeability / Class A/1 (Rhodopsin-like receptors) / 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 / response to acidic pH / 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 / G alpha (z) signalling events / Thrombin signalling through proteinase activated receptors (PARs) / Extra-nuclear estrogen signaling / G alpha (q) signalling events / G alpha (s) signalling events / photoreceptor outer segment membrane / spectrin binding / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / G alpha (i) signalling events / High laminar flow shear stress activates signaling by PIEZO1 and PECAM1:CDH5:KDR in endothelial cells / Vasopressin regulates renal water homeostasis via Aquaporins / sensory perception of taste / positive regulation of Rho protein signal transduction / alkylglycerophosphoethanolamine phosphodiesterase activity / retina development in camera-type eye / adenylate cyclase-activating G protein-coupled bile acid receptor signaling pathway / adenylate cyclase-activating serotonin receptor signaling pathway / regulation of skeletal muscle contraction / developmental growth / hair follicle placode formation / PKA activation in glucagon signalling / renal water homeostasis / cardiac muscle cell apoptotic process / regulation of cell adhesion / intracellular transport / photoreceptor outer segment / D1 dopamine receptor binding / vascular endothelial cell response to laminar fluid shear stress / Hedgehog 'off' state / activation of adenylate cyclase activity / adenylate cyclase-activating adrenergic receptor signaling pathway / cellular response to acidic pH / photoreceptor inner segment / cellular response to glucagon stimulus / intracellular glucose homeostasis / adenylate cyclase activator activity / positive regulation of insulin secretion involved in cellular response to glucose stimulus / trans-Golgi network membrane / positive regulation of insulin secretion / negative regulation of inflammatory response to antigenic stimulus / response to prostaglandin E / bone development / platelet aggregation / cognition / cell population proliferation / G protein-coupled receptor activity / G-protein beta/gamma-subunit complex binding / positive regulation of inflammatory response / phospholipase C-activating G protein-coupled receptor signaling pathway / sensory perception of smell / Glucagon signaling in metabolic regulation / Prostacyclin signalling through prostacyclin receptor / Glucagon-type ligand receptors / 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 / adenylate cyclase-activating dopamine receptor signaling pathway / cellular response to prostaglandin E stimulus
Similarity search - Function
G protein-coupled receptor 4 orphan / G-protein alpha subunit, group S / G protein alpha subunit, helical insertion / G protein alpha subunit / Guanine nucleotide binding protein (G-protein), alpha subunit / G-protein alpha subunit / G-alpha domain profile. / G-protein, gamma subunit / G-protein gamma subunit domain profile. / G-protein gamma-like domain ...G protein-coupled receptor 4 orphan / G-protein alpha subunit, group S / G protein alpha subunit, helical insertion / G protein alpha subunit / Guanine nucleotide binding protein (G-protein), alpha subunit / G-protein alpha subunit / G-alpha domain profile. / 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. / 7 transmembrane receptor (rhodopsin family) / G protein-coupled receptor, rhodopsin-like / GPCR, rhodopsin-like, 7TM / G-protein coupled receptors family 1 profile. / 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
CHOLESTEROL / G protein-coupled receptor 4 / Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Guanine nucleotide-binding protein G(s) subunit alpha isoforms short / Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
Homo sapiens (human)
Bos taurus (domestic cattle)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.4 Å
AuthorsChen, L.N. / Mao, C.Y. / Cheng, S.Z. / Liu, Y.F. / Fu, Y.F. / Ma, X.Y. / Xu, P. / Ji, S.Y. / Wang, W.W. / Shen, D.D. ...Chen, L.N. / Mao, C.Y. / Cheng, S.Z. / Liu, Y.F. / Fu, Y.F. / Ma, X.Y. / Xu, P. / Ji, S.Y. / Wang, W.W. / Shen, D.D. / Zhang, H.B. / Shen, Q.Y. / Chai, R. / Zhang, M. / Yang, L. / Han, F. / Mao, C.Y. / Cai, X.J. / Zhang, Y.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Mol Cell / Year: 2025
Title: Proton perception and activation of a proton-sensing GPCR.
Authors: Li-Nan Chen / Hui Zhou / Kun Xi / Shizhuo Cheng / Yongfeng Liu / Yifan Fu / Xiangyu Ma / Ping Xu / Su-Yu Ji / Wei-Wei Wang / Dan-Dan Shen / Huibing Zhang / Qingya Shen / Renjie Chai / Min ...Authors: Li-Nan Chen / Hui Zhou / Kun Xi / Shizhuo Cheng / Yongfeng Liu / Yifan Fu / Xiangyu Ma / Ping Xu / Su-Yu Ji / Wei-Wei Wang / Dan-Dan Shen / Huibing Zhang / Qingya Shen / Renjie Chai / Min Zhang / Lin Yang / Feng Han / Chunyou Mao / Xiujun Cai / Yan Zhang /
Abstract: Maintaining pH at cellular, tissular, and systemic levels is essential for human health. Proton-sensing GPCRs regulate physiological and pathological processes by sensing the extracellular acidity. ...Maintaining pH at cellular, tissular, and systemic levels is essential for human health. Proton-sensing GPCRs regulate physiological and pathological processes by sensing the extracellular acidity. However, the molecular mechanism of proton sensing and activation of these receptors remains elusive. Here, we present cryoelectron microscopy (cryo-EM) structures of human GPR4, a prototypical proton-sensing GPCR, in its inactive and active states. Our studies reveal that three extracellular histidine residues are crucial for proton sensing of human GPR4. The binding of protons induces substantial conformational changes in GPR4's ECLs, particularly in ECL2, which transforms from a helix-loop to a β-turn-β configuration. This transformation leads to the rearrangements of H-bond network and hydrophobic packing, relayed by non-canonical motifs to accommodate G proteins. Furthermore, the antagonist NE52-QQ57 hinders human GPR4 activation by preventing hydrophobic stacking rearrangement. Our findings provide a molecular framework for understanding the activation mechanism of a human proton-sensing GPCR, aiding future drug discovery.
History
DepositionApr 23, 2024Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Jul 16, 2025Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
C: Isoform Gnas-2 of Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
G: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
N: Nanobody-35
R: G-protein coupled receptor 4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)135,8377
Polymers135,0645
Non-polymers7732
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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Guanine nucleotide-binding protein ... , 2 types, 2 molecules BG

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


Mass: 37198.656 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Gnb1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P54311
#3: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / G gamma-I


Mass: 6261.229 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bos taurus (domestic cattle) / Gene: GNG2 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P63212

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Protein , 2 types, 2 molecules CR

#2: Protein Isoform Gnas-2 of Guanine nucleotide-binding protein G(s) subunit alpha isoforms short / Adenylate cyclase-stimulating G alpha protein


Mass: 43234.875 Da / Num. of mol.: 1 / Mutation: G226A,E268A,N271K,K274D,R280K,T284D,I271T
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNAS, GNAS1, GSP / Production host: Trichoplusia ni (cabbage looper)
References: UniProt: P63092, Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement
#5: Protein G-protein coupled receptor 4 / G-protein coupled receptor 6C.l / GPR6C.l


Mass: 34657.527 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GPR4 / Production host: Homo sapiens (human) / References: UniProt: P46093

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Antibody , 1 types, 1 molecules N

#4: Antibody Nanobody-35


Mass: 13711.284 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)

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Non-polymers , 2 types, 3 molecules

#6: Chemical ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: C27H46O
#7: Water ChemComp-HOH / 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
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: Cryo-EM structure of human GPR4-Gs complex / Type: COMPLEX / Entity ID: #1-#5 / Source: RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
31Bos taurus (domestic cattle)9913
41Rattus norvegicus (Norway rat)10116
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
31Escherichia coli (E. coli)562
41Trichoplusia ni (cabbage looper)7111
Buffer solutionpH: 6
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: 1500 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 52 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM software
IDNameCategory
1RELIONparticle selection
8PHENIXmodel refinement
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 158171 / Symmetry type: POINT
RefinementStereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0078523
ELECTRON MICROSCOPYf_angle_d0.82911556
ELECTRON MICROSCOPYf_dihedral_angle_d13.3445068
ELECTRON MICROSCOPYf_chiral_restr0.0491285
ELECTRON MICROSCOPYf_plane_restr0.0051470

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