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- PDB-8y6w: TUG-1375 and 4-CMTB-bound human FFA2 in complex with Gi -

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Basic information

Entry
Database: PDB / ID: 8y6w
TitleTUG-1375 and 4-CMTB-bound human FFA2 in complex with Gi
Components
  • (Guanine nucleotide-binding protein ...) x 3
  • Free fatty acid receptor 2
  • scFv16
KeywordsMEMBRANE PROTEIN / GPCR
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 / leukocyte chemotaxis involved in inflammatory response / mucosal immune 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 / leukocyte chemotaxis involved in inflammatory response / mucosal immune 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 / adenylate cyclase inhibitor activity / positive regulation of protein localization to cell cortex / T cell migration / Adenylate cyclase inhibitory pathway / D2 dopamine receptor binding / positive regulation of chemokine production / response to prostaglandin E / G protein-coupled serotonin receptor binding / adenylate cyclase regulator activity / adenylate cyclase-inhibiting serotonin receptor signaling pathway / cellular response to forskolin / regulation of mitotic spindle organization / cell projection / positive regulation of interleukin-8 production / Regulation of insulin secretion / positive regulation of cholesterol biosynthetic process / G protein-coupled receptor binding / negative regulation of insulin secretion / G protein-coupled receptor activity / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / positive regulation of insulin secretion / response to peptide hormone / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / G-protein beta/gamma-subunit complex binding / centriolar satellite / 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 / G-protein activation / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / 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 / Sensory perception of sweet, bitter, and umami (glutamate) taste / photoreceptor disc membrane / Glucagon-type ligand receptors / Adrenaline,noradrenaline inhibits insulin secretion / Vasopressin regulates renal water homeostasis via Aquaporins / GDP binding / G alpha (z) signalling events / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / cellular response to catecholamine stimulus / ADP signalling through P2Y purinoceptor 1 / ADORA2B mediated anti-inflammatory cytokines production / G beta:gamma signalling through PI3Kgamma / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / adenylate cyclase-activating dopamine receptor signaling pathway / GPER1 signaling / Inactivation, recovery and regulation of the phototransduction cascade / cellular response to prostaglandin E stimulus / glucose homeostasis / 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 / G protein activity / GTPase binding / Ca2+ pathway / midbody / fibroblast proliferation / cell cortex / 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 / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / G alpha (q) signalling events / Ras protein signal transduction / Extra-nuclear estrogen signaling / cell population proliferation / ciliary basal body / G protein-coupled receptor signaling pathway / lysosomal membrane / cell division / GTPase activity / synapse / lipid binding
Similarity search - Function
G protein-coupled receptor 40-related receptor / G-protein alpha subunit, group I / 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-coupled receptor 40-related receptor / G-protein alpha subunit, group I / 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 / 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 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Chem-9UJ / : / 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 / Guanine nucleotide-binding protein G(i) subunit alpha-1
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.19 Å
AuthorsKugawa, M. / Kawakami, K. / Kise, R. / Kobayashi, K. / Kojima, A. / Inoue, W. / Fukuda, M. / Inoue, A. / Kato, H.E.
Funding support Japan, 2items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)21H05142 Japan
Japan Society for the Promotion of Science (JSPS)21H04791 Japan
CitationJournal: Nat Commun / Year: 2025
Title: Structural insights into lipid chain-length selectivity and allosteric regulation of FFA2.
Authors: Mai Kugawa / Kouki Kawakami / Ryoji Kise / Carl-Mikael Suomivuori / Masaki Tsujimura / Kazuhiro Kobayashi / Asato Kojima / Wakana J Inoue / Masahiro Fukuda / Toshiki E Matsui / Ayami ...Authors: Mai Kugawa / Kouki Kawakami / Ryoji Kise / Carl-Mikael Suomivuori / Masaki Tsujimura / Kazuhiro Kobayashi / Asato Kojima / Wakana J Inoue / Masahiro Fukuda / Toshiki E Matsui / Ayami Fukunaga / Junki Koyanagi / Suhyang Kim / Hisako Ikeda / Keitaro Yamashita / Keisuke Saito / Hiroshi Ishikita / Ron O Dror / Asuka Inoue / Hideaki E Kato /
Abstract: The free fatty acid receptor 2 (FFA2) is a G protein-coupled receptor (GPCR) that selectively recognizes short-chain fatty acids to regulate metabolic and immune functions. As a promising therapeutic ...The free fatty acid receptor 2 (FFA2) is a G protein-coupled receptor (GPCR) that selectively recognizes short-chain fatty acids to regulate metabolic and immune functions. As a promising therapeutic target, FFA2 has been the focus of intensive development of synthetic ligands. However, the mechanisms by which endogenous and synthetic ligands modulate FFA2 activity remain unclear. Here, we present the structures of the human FFA2-Gi complex activated by the synthetic orthosteric agonist TUG-1375 and the positive allosteric modulator/allosteric agonist 4-CMTB, along with the structure of the inactive FFA2 bound to the antagonist GLPG0974. Structural comparisons with FFA1 and mutational studies reveal how FFA2 selects specific fatty acid chain lengths. Moreover, our structures reveal that GLPG0974 functions as an allosteric antagonist by binding adjacent to the orthosteric pocket to block agonist binding, whereas 4-CMTB binds the outer surface of transmembrane helices 6 and 7 to directly activate the receptor. Supported by computational and functional studies, these insights illuminate diverse mechanisms of ligand action, paving the way for precise GPCR-targeted drug design.
History
DepositionFeb 3, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 2, 2025Provider: repository / Type: Initial release
Revision 1.0Apr 2, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: 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(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
D: scFv16
R: Free fatty acid receptor 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)157,0067
Polymers156,2685
Non-polymers7382
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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

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


Mass: 40415.031 Da / Num. of mol.: 1
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
#2: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Transducin beta chain 1


Mass: 39286.891 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
#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 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P59768

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Antibody / Protein , 2 types, 2 molecules DR

#4: Antibody scFv16


Mass: 27784.896 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Trichoplusia ni (cabbage looper)
#5: Protein Free fatty acid receptor 2 / G-protein coupled receptor 43


Mass: 40919.961 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: Homo sapiens (human) / References: UniProt: O15552

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Non-polymers , 2 types, 2 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-A1LYC / (2~{S})-2-(4-chlorophenyl)-3-methyl-~{N}-(1,3-thiazol-2-yl)butanamide


Mass: 294.800 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C14H15ClN2OS / 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

Component
IDNameTypeEntity IDParent-IDSource
1TUG-1375 and 4-CMTB-bound human FFA2 in complex with GiCOMPLEX#1-#50MULTIPLE SOURCES
2Guanine nucleotide-binding protein G(i) subunit alpha-1COMPLEX#11RECOMBINANT
3Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1COMPLEX#21RECOMBINANT
4Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2COMPLEX#31RECOMBINANT
5scFv16COMPLEX#41RECOMBINANT
6Free fatty acid receptor 2COMPLEX#51RECOMBINANT
Molecular weight
IDEntity assembly-IDExperimental value
11NO
22
33
44
55
66
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
32Homo sapiens (human)9606
43Homo sapiens (human)9606
54Homo sapiens (human)9606
65Mus musculus (house mouse)10090
76Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
32Trichoplusia ni (cabbage looper)7111
43Trichoplusia ni (cabbage looper)7111
54Trichoplusia ni (cabbage looper)7111
65Trichoplusia ni (cabbage looper)7111
76Homo sapiens (human)9606
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: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1600 nm / Nominal defocus min: 800 nm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 45.579 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.19 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 256705 / Symmetry type: POINT
RefinementHighest resolution: 3.19 Å

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