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- PDB-7cx3: Cryo-EM structure of the Taprenepag-bound EP2-Gs complex -

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Basic information

Entry
Database: PDB / ID: 7cx3
TitleCryo-EM structure of the Taprenepag-bound EP2-Gs complex
Components
  • (Guanine nucleotide-binding protein ...) x 3
  • Nanobody-35
  • Prostaglandin E2 receptor EP2 subtype
KeywordsMEMBRANE PROTEIN / GPCR / EP2 / Complex / Taprenepag
Function / homology
Function and homology information


prostaglandin E receptor activity / Prostanoid ligand receptors / response to nematode / PKA activation in glucagon signalling / hair follicle placode formation / developmental growth / D1 dopamine receptor binding / intracellular transport / renal water homeostasis / Hedgehog 'off' state ...prostaglandin E receptor activity / Prostanoid ligand receptors / response to nematode / PKA activation in glucagon signalling / hair follicle placode formation / developmental growth / D1 dopamine receptor binding / intracellular transport / renal water homeostasis / Hedgehog 'off' state / adenylate cyclase-activating adrenergic receptor signaling pathway / activation of adenylate cyclase activity / cellular response to glucagon stimulus / adenylate cyclase activator activity / regulation of insulin secretion / trans-Golgi network membrane / response to progesterone / negative regulation of inflammatory response to antigenic stimulus / bone development / G-protein beta/gamma-subunit complex binding / Olfactory Signaling Pathway / Activation of the phototransduction cascade / adenylate cyclase-activating G protein-coupled receptor signaling pathway / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / G protein-coupled acetylcholine receptor signaling pathway / G protein activity / G-protein activation / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / platelet aggregation / Prostacyclin signalling through prostacyclin receptor / Glucagon signaling in metabolic regulation / G beta:gamma signalling through CDC42 / cognition / G beta:gamma signalling through BTK / ADP signalling through P2Y purinoceptor 12 / Sensory perception of sweet, bitter, and umami (glutamate) taste / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / 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 / ADORA2B mediated anti-inflammatory cytokines production / cellular response to catecholamine stimulus / sensory perception of taste / ADP signalling through P2Y purinoceptor 1 / G beta:gamma signalling through PI3Kgamma / adenylate cyclase-activating dopamine receptor signaling pathway / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / GPER1 signaling / cellular response to prostaglandin E stimulus / G-protein beta-subunit binding / Inactivation, recovery and regulation of the phototransduction cascade / heterotrimeric G-protein complex / sensory perception of smell / G alpha (12/13) signalling events / extracellular vesicle / signaling receptor complex adaptor activity / Thrombin signalling through proteinase activated receptors (PARs) / GTPase binding / positive regulation of cold-induced thermogenesis / retina development in camera-type eye / Ca2+ pathway / regulation of cell population proliferation / phospholipase C-activating G protein-coupled receptor signaling pathway / positive regulation of cytosolic calcium ion concentration / G alpha (i) signalling events / fibroblast proliferation / G alpha (s) signalling events / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / G alpha (q) signalling events / Ras protein signal transduction / response to lipopolysaccharide / cell population proliferation / Extra-nuclear estrogen signaling / 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 / cytosol / cytoplasm
Similarity search - Function
Prostanoid EP2 receptor / Prostanoid receptor / 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. ...Prostanoid EP2 receptor / Prostanoid receptor / 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. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD domain, G-beta repeat / 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
Chem-GNO / Prostaglandin E2 receptor EP2 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)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsQu, C. / Mao, C. / Xiao, P. / Shen, Q. / Zhong, Y. / Yang, F. / Shen, D. / Tao, X. / Zhang, H. / Yan, X. ...Qu, C. / Mao, C. / Xiao, P. / Shen, Q. / Zhong, Y. / Yang, F. / Shen, D. / Tao, X. / Zhang, H. / Yan, X. / Zhao, R. / He, J. / Guan, Y. / Zhang, C. / Hou, G. / Zhang, P. / Yu, X. / Guan, Y. / Sun, J. / Zhang, Y.
CitationJournal: Sci Adv / Year: 2021
Title: Ligand recognition, unconventional activation, and G protein coupling of the prostaglandin E receptor EP2 subtype.
Authors: Changxiu Qu / Chunyou Mao / Peng Xiao / Qingya Shen / Ya-Ni Zhong / Fan Yang / Dan-Dan Shen / Xiaona Tao / Huibing Zhang / Xu Yan / Ru-Jia Zhao / Junyan He / Ying Guan / Chao Zhang / Guihua ...Authors: Changxiu Qu / Chunyou Mao / Peng Xiao / Qingya Shen / Ya-Ni Zhong / Fan Yang / Dan-Dan Shen / Xiaona Tao / Huibing Zhang / Xu Yan / Ru-Jia Zhao / Junyan He / Ying Guan / Chao Zhang / Guihua Hou / Peng-Ju Zhang / Guige Hou / Zijian Li / Xiao Yu / Ren-Jie Chai / You-Fei Guan / Jin-Peng Sun / Yan Zhang /
Abstract: Selective modulation of the heterotrimeric G protein α S subunit-coupled prostaglandin E (PGE) receptor EP2 subtype is a promising therapeutic strategy for osteoporosis, ocular hypertension, ...Selective modulation of the heterotrimeric G protein α S subunit-coupled prostaglandin E (PGE) receptor EP2 subtype is a promising therapeutic strategy for osteoporosis, ocular hypertension, neurodegenerative diseases, and cardiovascular disorders. Here, we report the cryo-electron microscopy structure of the EP2-G complex with its endogenous agonist PGE and two synthesized agonists, taprenepag and evatanepag (CP-533536). These structures revealed distinct features of EP2 within the EP receptor family in terms of its unconventional receptor activation and G protein coupling mechanisms, including activation in the absence of a typical W "toggle switch" and coupling to G via helix 8. Moreover, inspection of the agonist-bound EP2 structures uncovered key motifs governing ligand selectivity. Our study provides important knowledge for agonist recognition and activation mechanisms of EP2 and will facilitate the rational design of drugs targeting the PGE signaling system.
History
DepositionSep 1, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0May 5, 2021Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
R: Prostaglandin E2 receptor EP2 subtype
A: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
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
N: Nanobody-35
hetero molecules


Theoretical massNumber of molelcules
Total (without water)147,1986
Polymers146,7205
Non-polymers4791
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area11780 Å2
ΔGint-61 kcal/mol
Surface area43660 Å2

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Components

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

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


Mass: 45683.434 Da / Num. of mol.: 1
Mutation: S54N, G226A, E268A, N271K, K274D, R280K, T284D, I285T
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNAS, GNAS1, GSP / Cell (production host): sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P63092
#3: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Transducin beta chain 1


Mass: 39489.160 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNB1 / Cell (production host): sf9 / 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 / Cell (production host): sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P59768

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

#1: Protein Prostaglandin E2 receptor EP2 subtype / PGE2 receptor EP2 subtype / Prostanoid EP2 receptor


Mass: 39800.430 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PTGER2 / Cell (production host): sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P43116
#5: Antibody Nanobody-35


Mass: 13885.439 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)
#6: Chemical ChemComp-GNO / 2-[3-[[(4-pyrazol-1-ylphenyl)methyl-pyridin-3-ylsulfonyl-amino]methyl]phenoxy]ethanoic acid


Mass: 478.520 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C24H22N4O5S / Feature type: SUBJECT OF INVESTIGATION

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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: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Taprenepag-bound EP2-Gs complexCOMPLEX#1-#50RECOMBINANT
2EP2COMPLEX#11RECOMBINANT
3Nb35COMPLEX#51RECOMBINANT
4GsCOMPLEX#2-#41RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23synthetic construct (others)32630
34Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCell
12Spodoptera frugiperda (fall armyworm)7108sf9
23Escherichia coli (E. coli)562
34Spodoptera frugiperda (fall armyworm)7108
Buffer solutionpH: 7.5
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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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 FIELD / Nominal magnification: 29000 X / Calibrated magnification: 49310 X / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 64 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 4785
Image scansMovie frames/image: 40

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Processing

SoftwareName: PHENIX / Version: 1.16_3549: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4Gctfv1.18CTF correction
7Coot0.89model fitting
9PHENIX1.16model refinement
13RELION3.0-beta23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3451005
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 243913 / Symmetry type: POINT
Atomic model buildingPDB-ID: 7CFM

7cfm

Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0038083
ELECTRON MICROSCOPYf_angle_d0.59710964
ELECTRON MICROSCOPYf_dihedral_angle_d17.0834809
ELECTRON MICROSCOPYf_chiral_restr0.1551247
ELECTRON MICROSCOPYf_plane_restr0.0041400

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