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- PDB-7s8o: CryoEM structure of Gi-coupled MRGPRX2 with small molecule agonis... -

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Basic information

Entry
Database: PDB / ID: 7s8o
TitleCryoEM structure of Gi-coupled MRGPRX2 with small molecule agonist (R)-Zinc-3573
Components
  • (Guanine nucleotide-binding protein ...) x 3
  • Soluble cytochrome b562,Mas-related G-protein coupled receptor member X2 chimera
  • scFv16
KeywordsSIGNALING PROTEIN / GPCR
Function / homology
Function and homology information


mast cell secretagogue receptor activity / mast cell activation / neuropeptide binding / Adenylate cyclase inhibitory pathway / PLC beta mediated events / positive regulation of protein localization to cell cortex / G protein-coupled serotonin receptor binding / cell cortex region / mast cell degranulation / sleep ...mast cell secretagogue receptor activity / mast cell activation / neuropeptide binding / Adenylate cyclase inhibitory pathway / PLC beta mediated events / positive regulation of protein localization to cell cortex / G protein-coupled serotonin receptor binding / cell cortex region / mast cell degranulation / sleep / Olfactory Signaling Pathway / Thromboxane signalling through TP receptor / regulation of cAMP-mediated signaling / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / positive regulation of cytokinesis / Activation of the phototransduction cascade / cellular response to forskolin / regulation of mitotic spindle organization / ADP signalling through P2Y purinoceptor 12 / Prostacyclin signalling through prostacyclin receptor / G-protein activation / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / negative regulation of synaptic transmission / G protein-coupled acetylcholine receptor signaling pathway / Adrenaline,noradrenaline inhibits insulin secretion / Glucagon signaling in metabolic regulation / cardiac muscle cell apoptotic process / G beta:gamma signalling through CDC42 / sensory perception of taste / alkylglycerophosphoethanolamine phosphodiesterase activity / G alpha (z) signalling events / G-protein beta/gamma-subunit complex / rhodopsin mediated signaling pathway / cellular response to catecholamine stimulus / G beta:gamma signalling through BTK / G-protein gamma-subunit binding / Glucagon-type ligand receptors / positive regulation of potassium ion transmembrane transport / Vasopressin regulates renal water homeostasis via Aquaporins / adenylate cyclase-activating dopamine receptor signaling pathway / GTPase activating protein binding / photoreceptor disc membrane / G-protein beta/gamma-subunit complex binding / photoreceptor outer segment membrane / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / spectrin binding / G-protein beta-subunit binding / cellular response to prostaglandin E stimulus / heterotrimeric G-protein complex / G beta:gamma signalling through PI3Kgamma / Regulation of insulin secretion / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / ADP signalling through P2Y purinoceptor 1 / G protein-coupled receptor activity / Wnt signaling pathway, calcium modulating pathway / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / Inactivation, recovery and regulation of the phototransduction cascade / G alpha (12/13) signalling events / ADORA2B mediated anti-inflammatory cytokines production / extracellular vesicle / response to peptide hormone / G protein-coupled receptor binding / sensory perception of pain / phospholipase C-activating G protein-coupled receptor signaling pathway / GTPase binding / retina development in camera-type eye / Ca2+ pathway / Thrombin signalling through proteinase activated receptors (PARs) / photoreceptor inner segment / G alpha (s) signalling events / G alpha (i) signalling events / GDP binding / G alpha (q) signalling events / negative regulation of inflammatory response to antigenic stimulus / cell population proliferation / midbody / cell body / Ras protein signal transduction / Extra-nuclear estrogen signaling / lysosomal membrane / cellular response to hypoxia / positive regulation of cytosolic calcium ion concentration / platelet activation / protein folding / periplasmic space / electron transfer activity / cell cycle / GTPase activity / G protein-coupled receptor signaling pathway / iron ion binding / centrosome / cell division / membrane raft / synapse / GTP binding / dendrite / protein-containing complex binding
Similarity search - Function
Mas-related G protein-coupled receptor X1/X2 / Mas-related G protein-coupled receptor family / G-protein alpha subunit, group I / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / G-protein gamma subunit domain profile. / G-protein, gamma subunit / G-protein gamma-like domain superfamily / G protein gamma subunit-like motifs ...Mas-related G protein-coupled receptor X1/X2 / Mas-related G protein-coupled receptor family / G-protein alpha subunit, group I / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / G-protein gamma subunit domain profile. / G-protein, gamma subunit / G-protein gamma-like domain superfamily / G protein gamma subunit-like motifs / GGL domain / G-protein gamma-like domain / Guanine nucleotide-binding protein, beta subunit / G-protein, beta subunit / 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 coupled receptors family 1 signature. / GPCR, rhodopsin-like, 7TM / G protein-coupled receptor, rhodopsin-like / 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 circular profile. / WD domain, G-beta repeat / Trp-Asp (WD) repeats 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
Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Guanine nucleotide-binding protein G(i) subunit alpha-1 / Chem-8IU / Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / Soluble cytochrome b562 / Mas-related G-protein coupled receptor member X2
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Homo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.58 Å
AuthorsCao, C. / Fay, J.F. / Gumpper, R.H. / Roth, B.L.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK) United States
CitationJournal: Nature / Year: 2021
Title: Structure, function and pharmacology of human itch GPCRs.
Authors: Can Cao / Hye Jin Kang / Isha Singh / He Chen / Chengwei Zhang / Wenlei Ye / Byron W Hayes / Jing Liu / Ryan H Gumpper / Brian J Bender / Samuel T Slocum / Brian E Krumm / Katherine Lansu / ...Authors: Can Cao / Hye Jin Kang / Isha Singh / He Chen / Chengwei Zhang / Wenlei Ye / Byron W Hayes / Jing Liu / Ryan H Gumpper / Brian J Bender / Samuel T Slocum / Brian E Krumm / Katherine Lansu / John D McCorvy / Wesley K Kroeze / Justin G English / Jeffrey F DiBerto / Reid H J Olsen / Xi-Ping Huang / Shicheng Zhang / Yongfeng Liu / Kuglae Kim / Joel Karpiak / Lily Y Jan / Soman N Abraham / Jian Jin / Brian K Shoichet / Jonathan F Fay / Bryan L Roth /
Abstract: The MRGPRX family of receptors (MRGPRX1-4) is a family of mas-related G-protein-coupled receptors that have evolved relatively recently. Of these, MRGPRX2 and MRGPRX4 are key physiological and ...The MRGPRX family of receptors (MRGPRX1-4) is a family of mas-related G-protein-coupled receptors that have evolved relatively recently. Of these, MRGPRX2 and MRGPRX4 are key physiological and pathological mediators of itch and related mast cell-mediated hypersensitivity reactions. MRGPRX2 couples to both G and G in mast cells. Here we describe agonist-stabilized structures of MRGPRX2 coupled to G and G in ternary complexes with the endogenous peptide cortistatin-14 and with a synthetic agonist probe, respectively, and the development of potent antagonist probes for MRGPRX2. We also describe a specific MRGPRX4 agonist and the structure of this agonist in a complex with MRGPRX4 and G. Together, these findings should accelerate the structure-guided discovery of therapeutic agents for pain, itch and mast cell-mediated hypersensitivity.
History
DepositionSep 18, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 17, 2021Provider: repository / Type: Initial release
Revision 1.1Dec 1, 2021Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID

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Structure visualization

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Assembly

Deposited unit
R: Soluble cytochrome b562,Mas-related G-protein coupled receptor member X2 chimera
B: Guanine nucleotide-binding protein G(i) subunit alpha-1
C: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
D: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
E: scFv16
hetero molecules


Theoretical massNumber of molelcules
Total (without water)170,7246
Polymers170,4175
Non-polymers3071
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area10280 Å2
ΔGint-68 kcal/mol
Surface area50280 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) subunit alpha-1 / Adenylate cyclase-inhibiting G alpha protein


Mass: 40414.047 Da / Num. of mol.: 1 / Mutation: S47N, G203A, E245A, A326S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNAI1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P63096
#3: 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: Trichoplusia ni (cabbage looper) / 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: Trichoplusia ni (cabbage looper) / References: UniProt: P59768

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

#1: Protein Soluble cytochrome b562,Mas-related G-protein coupled receptor member X2 chimera / Cytochrome b-562


Mass: 54054.625 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli), (gene. exp.) Homo sapiens (human)
Gene: cybC, MRGPRX2, MRGX2 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P0ABE7, UniProt: Q96LB1
#5: Antibody scFv16


Mass: 28668.922 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Spodoptera frugiperda (fall armyworm)
#6: Chemical ChemComp-8IU / (3R)-N,N-dimethyl-1-[(8S)-5-phenylpyrazolo[1,5-a]pyrimidin-7-yl]pyrrolidin-3-amine


Mass: 307.393 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C18H21N5 / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
Sequence detailsThe N-terminus of Soluble cytochrome b562, Mas-related G-protein coupled receptor member X2 chimera ...The N-terminus of Soluble cytochrome b562, Mas-related G-protein coupled receptor member X2 chimera contains FLAG- and HIS- tags annotated as expression tag, as well as an internal digestion site that is annotated as insertion.

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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: MRGPRX2-Gi1 (R)-ZINC-3573 / Type: COMPLEX / Entity ID: #1-#5 / Source: MULTIPLE SOURCES
Molecular weightValue: 150 kDa/nm / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Homo sapiens (human)9606
21Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
11Trichoplusia ni (cabbage looper)7111
21Spodoptera frugiperda (fall armyworm)7108
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 45 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameCategory
2SerialEMimage acquisition
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.58 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 705283 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0058508
ELECTRON MICROSCOPYf_angle_d0.68611561
ELECTRON MICROSCOPYf_dihedral_angle_d16.4561177
ELECTRON MICROSCOPYf_chiral_restr0.0461343
ELECTRON MICROSCOPYf_plane_restr0.0051450

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