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- PDB-8ihh: Cryo-EM structure of HCA2-Gi complex with LUF6283 -

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Entry
Database: PDB / ID: 8ihh
TitleCryo-EM structure of HCA2-Gi complex with LUF6283
Components
  • (Guanine nucleotide-binding protein ...) x 3
  • Soluble cytochrome b562,Hydroxycarboxylic acid receptor 2
  • scFv16
KeywordsSIGNALING PROTEIN / GPCR
Function / homology
Function and homology information


Activation of the phototransduction cascade / Olfactory Signaling Pathway / Sensory perception of sweet, bitter, and umami (glutamate) taste / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Prostacyclin signalling through prostacyclin receptor / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / G alpha (z) signalling events / Glucagon-type ligand receptors / G beta:gamma signalling through PI3Kgamma ...Activation of the phototransduction cascade / Olfactory Signaling Pathway / Sensory perception of sweet, bitter, and umami (glutamate) taste / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Prostacyclin signalling through prostacyclin receptor / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / G alpha (z) signalling events / Glucagon-type ligand receptors / G beta:gamma signalling through PI3Kgamma / G beta:gamma signalling through CDC42 / G beta:gamma signalling through BTK / Adrenaline,noradrenaline inhibits insulin secretion / ADP signalling through P2Y purinoceptor 12 / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / Thromboxane signalling through TP receptor / Thrombin signalling through proteinase activated receptors (PARs) / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / G-protein activation / G alpha (s) signalling events / nicotinic acid receptor activity / Hydroxycarboxylic acid-binding receptors / Ca2+ pathway / G alpha (12/13) signalling events / neutrophil apoptotic process / Extra-nuclear estrogen signaling / G alpha (q) signalling events / positive regulation of neutrophil apoptotic process / Vasopressin regulates renal water homeostasis via Aquaporins / GPER1 signaling / G alpha (i) signalling events / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / High laminar flow shear stress activates signaling by PIEZO1 and PECAM1:CDH5:KDR in endothelial cells / ADP signalling through P2Y purinoceptor 1 / phototransduction, visible light / Class A/1 (Rhodopsin-like receptors) / positive regulation of adiponectin secretion / spectrin binding / alkylglycerophosphoethanolamine phosphodiesterase activity / photoreceptor outer segment / negative regulation of lipid catabolic process / Adenylate cyclase inhibitory pathway / positive regulation of protein localization to cell cortex / T cell migration / D2 dopamine receptor binding / response to prostaglandin E / G protein-coupled serotonin receptor binding / adenylate cyclase-inhibiting serotonin receptor signaling pathway / adenylate cyclase regulator activity / regulation of mitotic spindle organization / cardiac muscle cell apoptotic process / cellular response to forskolin / Regulation of insulin secretion / G protein-coupled receptor binding / positive regulation of cholesterol biosynthetic process / electron transport chain / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / G-protein beta/gamma-subunit complex binding / response to peptide hormone / ADP signalling through P2Y purinoceptor 12 / Adrenaline,noradrenaline inhibits insulin secretion / G alpha (z) signalling events / ADORA2B mediated anti-inflammatory cytokines production / GDP binding / GPER1 signaling / G-protein beta-subunit binding / heterotrimeric G-protein complex / cell junction / sensory perception of taste / myelin sheath / G protein activity / GTPase binding / retina development in camera-type eye / cell cortex / midbody / fibroblast proliferation / phospholipase C-activating G protein-coupled receptor signaling pathway / G alpha (i) signalling events / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / G alpha (s) signalling events / cellular response to hypoxia / Extra-nuclear estrogen signaling / cell population proliferation / electron transfer activity / periplasmic space / G protein-coupled receptor signaling pathway / iron ion binding / lysosomal membrane / cell division / GTPase activity / centrosome / heme binding / synapse / protein-containing complex binding / nucleolus / GTP binding / magnesium ion binding / extracellular exosome
Similarity search - Function
: / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / G-protein alpha subunit, group I / YVTN repeat-like/Quinoprotein amine dehydrogenase / 7 Propeller / Methylamine Dehydrogenase; Chain H / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion ...: / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / G-protein alpha subunit, group I / YVTN repeat-like/Quinoprotein amine dehydrogenase / 7 Propeller / Methylamine Dehydrogenase; Chain H / 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 / Mainly Beta
Similarity search - Domain/homology
5-butyl-1~{H}-pyrazole-3-carboxylic acid / Soluble cytochrome b562 / Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Guanine nucleotide-binding protein G(i) subunit alpha-1 / Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / Hydroxycarboxylic acid receptor 2
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Homo sapiens (human)
Mus musculus (house mouse)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.06 Å
AuthorsSuzuki, S. / Nishikawa, K. / Suzuki, H. / Fujiyoshi, Y.
Funding support Japan, 1items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)20H00451 Japan
CitationJournal: Nat Commun / Year: 2023
Title: Structural basis of hydroxycarboxylic acid receptor signaling mechanisms through ligand binding.
Authors: Shota Suzuki / Kotaro Tanaka / Kouki Nishikawa / Hiroshi Suzuki / Atsunori Oshima / Yoshinori Fujiyoshi /
Abstract: Hydroxycarboxylic acid receptors (HCA) are expressed in various tissues and immune cells. HCA2 and its agonist are thus important targets for treating inflammatory and metabolic disorders. Only ...Hydroxycarboxylic acid receptors (HCA) are expressed in various tissues and immune cells. HCA2 and its agonist are thus important targets for treating inflammatory and metabolic disorders. Only limited information is available, however, on the active-state binding of HCAs with agonists. Here, we present cryo-EM structures of human HCA2-Gi and HCA3-Gi signaling complexes binding with multiple compounds bound. Agonists were revealed to form a salt bridge with arginine, which is conserved in the HCA family, to activate these receptors. Extracellular regions of the receptors form a lid-like structure that covers the ligand-binding pocket. Although transmembrane (TM) 6 in HCAs undergoes dynamic conformational changes, ligands do not directly interact with amino acids in TM6, suggesting that indirect signaling induces a slight shift in TM6 to activate Gi proteins. Structural analyses of agonist-bound HCA2 and HCA3 together with mutagenesis and molecular dynamics simulation provide molecular insights into HCA ligand recognition and activation mechanisms.
History
DepositionFeb 22, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Aug 30, 2023Provider: repository / Type: Initial release
Revision 1.1Dec 6, 2023Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Nov 6, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
R: Soluble cytochrome b562,Hydroxycarboxylic acid receptor 2
A: Guanine nucleotide-binding protein G(i) subunit alpha-1
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
S: scFv16
hetero molecules


Theoretical massNumber of molelcules
Total (without water)191,6847
Polymers191,2955
Non-polymers3892
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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

#2: Protein Guanine nucleotide-binding protein G(i) subunit alpha-1 / Adenylate cyclase-inhibiting G alpha protein


Mass: 40446.047 Da / Num. of mol.: 1 / Mutation: G203A, A326S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNAI1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P63096
#3: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1


Mass: 41772.562 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Gnb1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P62874
#4: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / G gamma-I


Mass: 7729.947 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Gng2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P63213

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Protein / Antibody / Sugars / Non-polymers , 4 types, 4 molecules RS

#1: Protein Soluble cytochrome b562,Hydroxycarboxylic acid receptor 2 / Cytochrome b-562 / G-protein coupled receptor 109A / G-protein coupled receptor HM74A / Niacin ...Cytochrome b-562 / G-protein coupled receptor 109A / G-protein coupled receptor HM74A / Niacin receptor 1 / Nicotinic acid receptor


Mass: 74879.688 Da / Num. of mol.: 1 / Mutation: M29W,H124I
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli), (gene. exp.) Homo sapiens (human)
Gene: cybC, HCAR2, GPR109A, HCA2, HM74A, NIACR1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P0ABE7, UniProt: Q8TDS4
#5: Antibody scFv16


Mass: 26466.486 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Spodoptera frugiperda (fall armyworm)
#6: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#7: Chemical ChemComp-P8A / 5-butyl-1~{H}-pyrazole-3-carboxylic acid


Mass: 168.193 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H12N2O2 / 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: Multiprotein complex / Type: COMPLEX / Entity ID: #2-#4, #1, #5 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.4
SpecimenConc.: 15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 49 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.06 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 181273 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0028874
ELECTRON MICROSCOPYf_angle_d0.47812050
ELECTRON MICROSCOPYf_dihedral_angle_d4.4611226
ELECTRON MICROSCOPYf_chiral_restr0.041377
ELECTRON MICROSCOPYf_plane_restr0.0041531

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