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- PDB-9wp9: Cryo-EM structure of the d18:1 S1P-bound S1PR3 and Gq complex -

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Basic information

Entry
Database: PDB / ID: 9wp9
TitleCryo-EM structure of the d18:1 S1P-bound S1PR3 and Gq complex
Components
  • (Guanine nucleotide-binding protein ...) x 2
  • Engineered G-alpha-q
  • Nb35
  • Sphingosine 1-phosphate receptor 3
  • scFv16
KeywordsMEMBRANE PROTEIN / GPCR / SBDD / lipid
Function / homology
Function and homology information


negative regulation of establishment of endothelial barrier / sphingosine-1-phosphate receptor activity / Lysosphingolipid and LPA receptors / regulation of interleukin-1 beta production / regulation of metabolic process / anatomical structure morphogenesis / Notch signaling pathway / G protein-coupled receptor activity / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / integrin binding ...negative regulation of establishment of endothelial barrier / sphingosine-1-phosphate receptor activity / Lysosphingolipid and LPA receptors / regulation of interleukin-1 beta production / regulation of metabolic process / anatomical structure morphogenesis / Notch signaling pathway / G protein-coupled receptor activity / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / integrin binding / adenylate cyclase-activating G protein-coupled receptor signaling pathway / Olfactory Signaling Pathway / 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 / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / G-protein activation / 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 / photoreceptor disc membrane / Sensory perception of sweet, bitter, and umami (glutamate) taste / Glucagon-type ligand receptors / Adrenaline,noradrenaline inhibits insulin secretion / Vasopressin regulates renal water homeostasis via Aquaporins / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / G alpha (z) signalling events / 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 / 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 / presynapse / sensory perception of taste / extracellular vesicle / signaling receptor complex adaptor activity / Thrombin signalling through proteinase activated receptors (PARs) / retina development in camera-type eye / positive regulation of cytosolic calcium ion concentration / GTPase binding / Ca2+ pathway / fibroblast proliferation / High laminar flow shear stress activates signaling by PIEZO1 and PECAM1:CDH5:KDR in endothelial cells / G alpha (i) signalling events / 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 / inflammatory response / lysosomal membrane / GTPase activity / positive regulation of cell population proliferation / synapse / lipid binding / protein-containing complex binding / signal transduction / extracellular exosome / membrane / plasma membrane / cytosol / cytoplasm
Similarity search - Function
EDG-3 sphingosine 1-phosphate receptor / Sphingosine 1-phosphate receptor / Serpentine type 7TM GPCR chemoreceptor Srsx / 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 ...EDG-3 sphingosine 1-phosphate receptor / Sphingosine 1-phosphate receptor / Serpentine type 7TM GPCR chemoreceptor Srsx / 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
Similarity search - Domain/homology
Chem-S1P / 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 / Sphingosine 1-phosphate receptor 3
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.25 Å
AuthorsIm, D. / Asada, H. / Iwata, S. / Yamauchi, M. / Hagiwara, M.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Proc Natl Acad Sci U S A / Year: 2025
Title: Structural insights into the G-protein subtype selectivity revealed by human sphingosine-1-phosphate receptor 3-G complexes.
Authors: Momono Yamauchi / Dohyun Im / Shintaro Maeda / Tatsuya Ikuta / Masayasu Toyomoto / Hidetsugu Asada / Yukihiko Sugita / Jun-Ichi Kishikawa / Takeshi Noda / Takayuki Kato / Asuka Inoue / So ...Authors: Momono Yamauchi / Dohyun Im / Shintaro Maeda / Tatsuya Ikuta / Masayasu Toyomoto / Hidetsugu Asada / Yukihiko Sugita / Jun-Ichi Kishikawa / Takeshi Noda / Takayuki Kato / Asuka Inoue / So Iwata / Masatoshi Hagiwara /
Abstract: Sphingosine-1-phosphate (S1P) is one of the most extensively studied bioactive lipids that transduces signals via the S1P receptor (S1PR) family (S1PR1-5), a class of G-protein-coupled receptors ...Sphingosine-1-phosphate (S1P) is one of the most extensively studied bioactive lipids that transduces signals via the S1P receptor (S1PR) family (S1PR1-5), a class of G-protein-coupled receptors (GPCRs), to regulate immune cell migration, vascular permeability, and pain modulation. However, the mechanism for achieving specificity in downstream signaling remains poorly understood. Here, we present cryogenic electron microscopic structures of the S1PR3-G complex bound to endogenous agonists: d18:1 S1P or d16:1 S1P. Both agonists shared the same binding pocket and binding mode despite the different signaling intensities of the S1PR3-G signal pathway. By comparing the structures of two agonist-bound complexes, combined with mutagenesis studies, we identified key amino acids, Phe119 and Arg136, that play crucial roles in differential agonist recognition and receptor activation. Furthermore, structural comparisons with previously determined S1PR3-G complex or G-protein-free S1PR3 structures, along with mutagenesis analysis, revealed dynamic intracellular loop 2 conformations and specific amino acid interactions that contribute to G-protein selectivity. Notably, we identified amino acids at the 34.50 and 34.53 positions within ICL2 as critical for specific interactions with G proteins. These findings provide better understanding of the mechanism of GPCR activation and unique perspectives that can be applied to other class A GPCRs, leading to the possibility of optimized drug development.
History
DepositionSep 8, 2025Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 26, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Engineered G-alpha-q
B: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
C: scFv16
G: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
N: Nb35
R: Sphingosine 1-phosphate receptor 3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)155,9707
Polymers155,5906
Non-polymers3791
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 3 types, 3 molecules ANR

#1: Protein Engineered G-alpha-q


Mass: 27680.387 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Spodoptera frugiperda (fall armyworm)
#5: Protein Nb35


Mass: 14016.636 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Spodoptera frugiperda (fall armyworm)
#6: Protein Sphingosine 1-phosphate receptor 3 / S1P receptor 3 / S1P3 / Endothelial differentiation G-protein coupled receptor 3 / Sphingosine 1- ...S1P receptor 3 / S1P3 / Endothelial differentiation G-protein coupled receptor 3 / Sphingosine 1-phosphate receptor Edg-3 / S1P receptor Edg-3


Mass: 38829.141 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: S1PR3, C9orf108, C9orf47, EDG3 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q99500

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Guanine nucleotide-binding protein ... , 2 types, 2 molecules BG

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


Mass: 39418.086 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNB1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P62873
#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.) Homo sapiens (human) / Gene: GNG2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P59768

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Antibody / Non-polymers , 2 types, 2 molecules C

#3: Antibody scFv16


Mass: 27784.896 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Spodoptera frugiperda (fall armyworm)
#7: Chemical ChemComp-S1P / (2S,3R,4E)-2-amino-3-hydroxyoctadec-4-en-1-yl dihydrogen phosphate / sphingosine 1-phosphate


Mass: 379.472 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C18H38NO5P / Feature type: SUBJECT OF INVESTIGATION

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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: d18:1 S1P-bound S1PR3 in complex with Gq / Type: COMPLEX / Entity ID: #1-#6 / Source: RECOMBINANT
Molecular weightValue: 0.19 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
SpecimenConc.: 10 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281.15 K

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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: 1800 nm / Nominal defocus min: 800 nm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2PHENIX1.20.1_4487model refinement
13cryoSPARC3D reconstruction
CTF correctionType: NONE
3D reconstructionResolution: 3.25 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 366554 / Symmetry type: POINT
RefinementHighest resolution: 3.25 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0039945
ELECTRON MICROSCOPYf_angle_d0.66313460
ELECTRON MICROSCOPYf_dihedral_angle_d4.7841371
ELECTRON MICROSCOPYf_chiral_restr0.0451524
ELECTRON MICROSCOPYf_plane_restr0.0041702

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