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- PDB-9cm3: Cryo-EM structure of Gq-coupled FFA2 in complex with TUG-1375 and... -

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Basic information

Entry
Database: PDB / ID: 9cm3
TitleCryo-EM structure of Gq-coupled FFA2 in complex with TUG-1375 and compound 187
Components
  • (Guanine nucleotide-binding protein ...) x 3
  • Free fatty acid receptor 2
  • scFv16
KeywordsMEMBRANE PROTEIN / GPCR / agonist
Function / homology
Function and homology information


positive regulation of acute inflammatory response to non-antigenic stimulus / regulation of peptide hormone secretion / regulation of acute inflammatory response / mucosal immune response / leukocyte chemotaxis involved in inflammatory response / Free fatty acid receptors / positive regulation of cytokine production involved in immune response / cell surface pattern recognition receptor signaling pathway / lipid storage / cellular response to fatty acid ...positive regulation of acute inflammatory response to non-antigenic stimulus / regulation of peptide hormone secretion / regulation of acute inflammatory response / mucosal immune response / leukocyte chemotaxis involved in inflammatory response / Free fatty acid receptors / positive regulation of cytokine production involved in immune response / cell surface pattern recognition receptor signaling pathway / lipid storage / cellular response to fatty acid / fat cell differentiation / ligand-gated ion channel signaling pathway / positive regulation of chemokine production / positive regulation of interleukin-8 production / cell projection / negative regulation of insulin secretion / G protein-coupled receptor activity / positive regulation of insulin secretion / 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 / ADP signalling through P2Y purinoceptor 1 / ADORA2B mediated anti-inflammatory cytokines production / 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 / Inactivation, recovery and regulation of the phototransduction cascade / glucose homeostasis / cellular response to prostaglandin E stimulus / G-protein beta-subunit binding / heterotrimeric G-protein complex / G alpha (12/13) signalling events / sensory perception of taste / extracellular vesicle / signaling receptor complex adaptor activity / Thrombin signalling through proteinase activated receptors (PARs) / retina development in camera-type eye / 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 / lysosomal membrane / GTPase activity / synapse / lipid binding / protein-containing complex binding / signal transduction / extracellular exosome / membrane / plasma membrane / cytoplasm / cytosol
Similarity search - Function
G protein-coupled receptor 40-related receptor / 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-coupled receptor 40-related receptor / 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-9UJ / : / CHOLESTEROL / PALMITIC ACID / Free fatty acid receptor 2 / 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
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.06 Å
AuthorsZhang, X. / Tikhonova, I. / Milligan, G. / Zhang, C.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM128641 United States
CitationJournal: Nature / Year: 2025
Title: Allosteric modulation and biased signalling at free fatty acid receptor 2.
Authors: Xuan Zhang / Abdul-Akim Guseinov / Laura Jenkins / Alice Valentini / Sara Marsango / Katrine Schultz-Knudsen / Trond Ulven / Elisabeth Rexen Ulven / Irina G Tikhonova / Graeme Milligan / Cheng Zhang /
Abstract: Free fatty acid receptor 2 (FFA2) is a G protein-coupled receptor (GPCR) that is a primary sensor for short-chain fatty acids produced by gut microbiota. Consequently, FFA2 is a promising drug ...Free fatty acid receptor 2 (FFA2) is a G protein-coupled receptor (GPCR) that is a primary sensor for short-chain fatty acids produced by gut microbiota. Consequently, FFA2 is a promising drug target for immunometabolic disorders. Here we report cryogenic electronic microscopy structures of FFA2 in complex with two G proteins and three distinct classes of positive allosteric modulators (PAMs), and describe noncanonical activation mechanisms that involve conserved structural features of class A GPCRs. Two PAMs disrupt the E/DRY activation microswitch and stabilize the conformation of intracellular loop 2 by binding to lipid-facing pockets near the cytoplasmic side of the receptor. By contrast, the third PAM promotes the separation of transmembrane helices 6 and 7 by interacting with transmembrane helix 6 at the receptor-lipid interface. Molecular dynamic simulations and mutagenesis experiments confirm these noncanonical activation mechanisms. Furthermore, we demonstrate the molecular basis for the G versus G bias, which is due to distinct conformations of intracellular loop 2 stabilized by different PAMs. These findings provide a framework for the design of tailored GPCR modulators, with implications that extend beyond FFA2 to the broader field of GPCR drug discovery.
History
DepositionJul 12, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 25, 2025Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Guanine nucleotide-binding protein G(q) subunit alpha
B: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
N: scFv16
R: Free fatty acid receptor 2
G: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)167,9559
Polymers166,4495
Non-polymers1,5054
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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

#1: Protein Guanine nucleotide-binding protein G(q) subunit alpha


Mass: 28084.832 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNAQ / Production host: Spodoptera frugiperda (fall armyworm)
#2: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1


Mass: 40845.559 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
#5: 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 / Protein , 2 types, 2 molecules NR

#3: Antibody scFv16


Mass: 28634.797 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)
#4: Protein Free fatty acid receptor 2 / G-protein coupled receptor 43


Mass: 61023.004 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: FFAR2, FFA2, GPCR43, GPR43 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: O15552

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Non-polymers , 4 types, 4 molecules

#6: Chemical ChemComp-9UJ / (2R,4R)-2-(2-chlorophenyl)-3-[4-(3,5-dimethyl-1,2-oxazol-4-yl)phenyl]carbonyl-1,3-thiazolidine-4-carboxylic acid / TUG-1375


Mass: 442.915 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C22H19ClN2O4S / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-A1AZC / 4-[(2R,6S)-2,6-dimethylmorpholin-4-yl]-7-(2-fluorobenzene-1-sulfonyl)-2-methyl-5H-pyrrolo[3,2-d]pyrimidin-6-amine


Mass: 419.473 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C19H22FN5O3S / Feature type: SUBJECT OF INVESTIGATION
#8: Chemical ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C27H46O / Feature type: SUBJECT OF INVESTIGATION
#9: Chemical ChemComp-PLM / PALMITIC ACID


Mass: 256.424 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C16H32O2 / 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: TUG-1375-compound 187-FFA2-miniGq complex / Type: COMPLEX / Entity ID: #1-#5 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 55 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
8PHENIXmodel refinement
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.06 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 348210 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0029041
ELECTRON MICROSCOPYf_angle_d0.43812286
ELECTRON MICROSCOPYf_dihedral_angle_d5.6911239
ELECTRON MICROSCOPYf_chiral_restr0.0391389
ELECTRON MICROSCOPYf_plane_restr0.0031558

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