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- PDB-7d7m: Cryo-EM Structure of the Prostaglandin E Receptor EP4 Coupled to ... -

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Basic information

Entry
Database: PDB / ID: 7d7m
TitleCryo-EM Structure of the Prostaglandin E Receptor EP4 Coupled to G Protein
Components
  • (Guanine nucleotide-binding protein ...) x 3
  • Prostaglandin E2 receptor EP4 subtype,Prostaglandin E2 receptor EP4 subtype
  • nanobody Nb35
KeywordsMEMBRANE PROTEIN / prostaglandin E receptor / EP4 / GPCR / G protein
Function / homology
Function and homology information


negative regulation of eosinophil extravasation / prostaglandin E receptor activity / Prostanoid ligand receptors / negative regulation of integrin activation / response to nematode / T-helper cell differentiation / regulation of stress fiber assembly / negative regulation of cytokine production / PKA activation in glucagon signalling / hair follicle placode formation ...negative regulation of eosinophil extravasation / prostaglandin E receptor activity / Prostanoid ligand receptors / negative regulation of integrin activation / response to nematode / T-helper cell differentiation / regulation of stress fiber assembly / negative regulation of cytokine production / PKA activation in glucagon signalling / hair follicle placode formation / intracellular transport / D1 dopamine receptor binding / developmental growth / regulation of ossification / Hedgehog 'off' state / positive regulation of cAMP-mediated signaling / response to mechanical stimulus / adenylate cyclase-activating adrenergic receptor signaling pathway / JNK cascade / ERK1 and ERK2 cascade / activation of adenylate cyclase activity / adenylate cyclase activator activity / trans-Golgi network membrane / positive regulation of cytokine production / G-protein beta/gamma-subunit complex binding / 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 / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / bone development / G-protein activation / G protein-coupled acetylcholine receptor signaling pathway / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / Prostacyclin signalling through prostacyclin receptor / Glucagon signaling in metabolic regulation / G beta:gamma signalling through CDC42 / adenylate cyclase-activating G protein-coupled receptor signaling pathway / ADP signalling through P2Y purinoceptor 12 / G beta:gamma signalling through BTK / Sensory perception of sweet, bitter, and umami (glutamate) taste / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / photoreceptor disc membrane / Adrenaline,noradrenaline inhibits insulin secretion / platelet aggregation / negative regulation of inflammatory response / Glucagon-type ligand receptors / cognition / Vasopressin regulates renal water homeostasis via Aquaporins / positive regulation of GTPase activity / G alpha (z) signalling events / cellular response to catecholamine stimulus / positive regulation of inflammatory response / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / ADORA2B mediated anti-inflammatory cytokines production / adenylate cyclase-activating dopamine receptor signaling pathway / ADP signalling through P2Y purinoceptor 1 / G beta:gamma signalling through PI3Kgamma / cellular response to mechanical stimulus / cellular response to prostaglandin E stimulus / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / sensory perception of taste / GPER1 signaling / G-protein beta-subunit binding / heterotrimeric G-protein complex / Inactivation, recovery and regulation of the phototransduction cascade / extracellular vesicle / G alpha (12/13) signalling events / signaling receptor complex adaptor activity / sensory perception of smell / Thrombin signalling through proteinase activated receptors (PARs) / retina development in camera-type eye / GTPase binding / Ca2+ pathway / phospholipase C-activating G protein-coupled receptor signaling pathway / positive regulation of cold-induced thermogenesis / positive regulation of cytosolic calcium ion concentration / G alpha (i) signalling events / fibroblast proliferation / G alpha (s) signalling events / G alpha (q) signalling events / Ras protein signal transduction / cell population proliferation / response to lipopolysaccharide / Extra-nuclear estrogen signaling / immune response / inflammatory response / G protein-coupled receptor signaling pathway / lysosomal membrane / GTPase activity / synapse / protein-containing complex binding / GTP binding / signal transduction / extracellular exosome / membrane / metal ion binding / plasma membrane
Similarity search - Function
Prostanoid EP4 receptor / Prostaglandin DP receptor / Prostanoid receptor / G-protein alpha subunit, group S / G-alpha domain profile. / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G protein alpha subunit / G-protein, gamma subunit ...Prostanoid EP4 receptor / Prostaglandin DP receptor / Prostanoid receptor / G-protein alpha subunit, group S / G-alpha domain profile. / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G protein alpha subunit / G-protein, gamma subunit / G-protein gamma subunit domain profile. / GGL domain / G-protein gamma-like domain superfamily / G-protein gamma-like domain / GGL domain / G protein gamma subunit-like motifs / 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 / WD40 repeats / WD40 repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Chem-P2E / Prostaglandin E2 receptor EP4 subtype / 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)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsNojima, S. / Fujita, Y. / Kimura, T.K. / Nomura, N. / Suno, R. / Morimoto, K. / Yamamoto, M. / Noda, T. / Iwata, S. / Shigematsu, H. / Kobayashi, T.
Funding support Japan, 5items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)JP19J12880 Japan
Japan Agency for Medical Research and Development (AMED)JP20gm0910007 Japan
Japan Agency for Medical Research and Development (AMED)JP20am0401020 Japan
Japan Agency for Medical Research and Development (AMED)JP20ak0101103 Japan
Japan Agency for Medical Research and Development (AMED)JP19am0101115 Japan
CitationJournal: Structure / Year: 2021
Title: Cryo-EM Structure of the Prostaglandin E Receptor EP4 Coupled to G Protein.
Authors: Shingo Nojima / Yoko Fujita / Kanako Terakado Kimura / Norimichi Nomura / Ryoji Suno / Kazushi Morimoto / Masaki Yamamoto / Takeshi Noda / So Iwata / Hideki Shigematsu / Takuya Kobayashi /
Abstract: Prostaglandin E receptor EP4, a class A G protein-coupled receptor (GPCR), is a common drug target in various disorders, such as acute decompensated heart failure and ulcerative colitis. Here, we ...Prostaglandin E receptor EP4, a class A G protein-coupled receptor (GPCR), is a common drug target in various disorders, such as acute decompensated heart failure and ulcerative colitis. Here, we report the cryoelectron microscopy (cryo-EM) structure of the EP4-heterotrimeric G protein (Gs) complex with the endogenous ligand at a global resolution of 3.3 Å. In this structure, compared with that in the inactive EP4 structure, the sixth transmembrane domain is shifted outward on the intracellular side, although the shift is smaller than that in other class A GPCRs bound to Gs. Instead, the C-terminal helix of Gs is inserted toward TM2 of EP4, and the conserved C-terminal hook structure formsthe extended state. These structural features are formed by the conserved residues in prostanoid receptors (Phe54 and Trp327). These findings may be important for the thorough understanding of the G protein-binding mechanism of EP4 and other prostanoid receptors.
History
DepositionOct 5, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 18, 2020Provider: repository / Type: Initial release
Revision 2.0Dec 2, 2020Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Database references / Derived calculations / Structure summary
Category: atom_site / atom_site_anisotrop ...atom_site / atom_site_anisotrop / em_software / entity / pdbx_nonpoly_scheme / pdbx_poly_seq_scheme / pdbx_struct_sheet_hbond / pdbx_unobs_or_zero_occ_atoms / pdbx_unobs_or_zero_occ_residues / pdbx_validate_torsion / struct_conf / struct_ref_seq / struct_ref_seq_dif / struct_sheet_range
Item: _atom_site_anisotrop.U[1][2] / _atom_site_anisotrop.U[1][3] ..._atom_site_anisotrop.U[1][2] / _atom_site_anisotrop.U[1][3] / _atom_site_anisotrop.U[2][3] / _atom_site_anisotrop.id / _atom_site_anisotrop.pdbx_auth_seq_id / _em_software.category / _em_software.fitting_id / _em_software.imaging_id / _em_software.name / _em_software.version / _entity.details / _pdbx_nonpoly_scheme.auth_seq_num / _pdbx_poly_seq_scheme.auth_seq_num / _pdbx_poly_seq_scheme.pdb_seq_num / _pdbx_struct_sheet_hbond.range_1_auth_seq_id / _pdbx_struct_sheet_hbond.range_2_auth_seq_id / _pdbx_unobs_or_zero_occ_atoms.auth_seq_id / _pdbx_unobs_or_zero_occ_residues.auth_seq_id / _pdbx_validate_torsion.auth_seq_id / _struct_conf.beg_auth_seq_id / _struct_conf.end_auth_seq_id / _struct_ref_seq.pdbx_auth_seq_align_beg / _struct_ref_seq.pdbx_auth_seq_align_end / _struct_ref_seq_dif.pdbx_auth_seq_num / _struct_sheet_range.beg_auth_seq_id / _struct_sheet_range.end_auth_seq_id
Description: Atoms with unrealistic or zero occupancies
Details: We removed H atoms from the previous coordinates because the density of them were not observed in the EM map. We also adjusted the residue numbers of some chains for the sequence of the wild ...Details: We removed H atoms from the previous coordinates because the density of them were not observed in the EM map. We also adjusted the residue numbers of some chains for the sequence of the wild type so that the readers of our paper will refer the coordinate file easily.
Provider: author / Type: Coordinate replacement
Revision 2.1Dec 16, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.name
Revision 2.2Mar 17, 2021Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
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Assembly

Deposited unit
A: Prostaglandin E2 receptor EP4 subtype,Prostaglandin E2 receptor EP4 subtype
B: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
C: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
D: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short,Guanine nucleotide-binding protein G(s) subunit alpha isoforms short,Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
E: nanobody Nb35
hetero molecules


Theoretical massNumber of molelcules
Total (without water)126,5906
Polymers126,2385
Non-polymers3521
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area10500 Å2
ΔGint-73 kcal/mol
Surface area42730 Å2

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Components

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

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


Mass: 37728.152 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNB1 / Plasmid: pFastBac Dual / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P62873
#3: 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 / Plasmid: pFastBac Dual / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P59768
#4: Protein Guanine nucleotide-binding protein G(s) subunit alpha isoforms short,Guanine nucleotide-binding protein G(s) subunit alpha isoforms short,Guanine nucleotide-binding protein G(s) subunit alpha isoforms short / Adenylate cyclase-stimulating G alpha protein


Mass: 29014.750 Da / Num. of mol.: 1
Mutation: 65 to 203, 255 to 264 deletion G49D, E50N, L63Y, A249D, S252D, I372A and V375I
Source method: isolated from a genetically manipulated source
Details: residues from 65 to 203, residues from 255 to 264 were deleted
Source: (gene. exp.) Homo sapiens (human) / Gene: GNAS, GNAS1, GSP / Plasmid: pET21a / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P63092

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Protein / Antibody / Non-polymers , 3 types, 3 molecules AE

#1: Protein Prostaglandin E2 receptor EP4 subtype,Prostaglandin E2 receptor EP4 subtype / PGE2 receptor EP4 subtype / Prostanoid EP4 receptor


Mass: 36953.453 Da / Num. of mol.: 1 / Mutation: N7Q, N177Q, 218-259 deletion
Source method: isolated from a genetically manipulated source
Details: residues from 218 to 259 were deleted / Source: (gene. exp.) Homo sapiens (human) / Gene: PTGER4, PTGER2 / Plasmid: pFastBac1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P35408
#5: Antibody nanobody Nb35


Mass: 14680.293 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: The C-terminal "ENLYFQ" of sample sequence is a cleavaged TEV protease recognition sequence.
Source: (gene. exp.) Lama glama (llama) / Plasmid: pNY326 / Production host: Brevibacillus choshinensis (bacteria)
#6: Chemical ChemComp-P2E / (Z)-7-[(1R,2R,3R)-3-hydroxy-2-[(E,3S)-3-hydroxyoct-1-enyl]-5-oxo-cyclopentyl]hept-5-enoic acid / Prostaglandin E2 / Prostaglandin E2


Mass: 352.465 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C20H32O5 / Feature type: SUBJECT OF INVESTIGATION / Comment: medication*YM

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: 2D ARRAY / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1PGE2-EP4-Gs-Gbeta1-Ggamma2-Nb35 complexCOMPLEX#1-#50MULTIPLE SOURCES
2Prostaglandin E2 receptor EP4 subtype,Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1,Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2COMPLEX#1-#31RECOMBINANT
3Guanine nucleotide-binding protein G(s) subunit alpha isoforms shortCOMPLEX#41RECOMBINANT
4nanobody Nb35COMPLEX#51RECOMBINANT
Molecular weightValue: 0.12 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Homo sapiens (human)9606
34Lama glama (llama)9844
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Spodoptera frugiperda (fall armyworm)7108
23Escherichia coli BL21(DE3) (bacteria)469008
34Brevibacillus choshinensis (bacteria)54911
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMHEPES1
2100 mMsodium chlorideNaClSodium chloride1
30.00075 %lauryl maltose neopentyl glycol1
40.00025 %GDN1
50.000125 %cholesteryl hemisuccinate1
60.01 mMprostaglandin E 21
SpecimenConc.: 8.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 10 mA / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R0.6/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K / Details: Blot Force 10, Blot Time 3.5 sec, 3 microL apply

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Residual tilt: 0.01 mradians
Image recordingAverage exposure time: 1.83 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5743
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 25 eV
Image scansSampling size: 5 µm / Width: 4092 / Height: 5760

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Processing

EM software
IDNameVersionCategoryDetails
2SerialEM3.8.0BETAimage acquisitionOriginal script by Rado Danev
4CTFFIND4CTF correction
7Coot0.8.9.2model fitting
9PHENIX1.18-3845model refinement
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
13RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 2796263
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 178217 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-ID
15YWY

5ywy

A1
26GDG

6gdg

B1
36GDG

6gdg

C1
46GDG

6gdg

D1
56GDG

6gdg

E1

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