Molecular mechanism of fatty acid activation of FFAR1

Abstract

FFAR1 is a G-protein-coupled receptor (GPCR) that responds to circulating free fatty acids to enhance glucose-stimulated insulin secretion and release of incretin hormones. Due to the glucose-lowering effect of FFAR1 activation, potent agonists for this receptor have been developed for the treatment of diabetes. Previous structural and biochemical studies of FFAR1 showed multiple sites of ligand binding to the inactive state but left the mechanism of fatty acid interaction and receptor activation unknown. We used cryo-electron microscopy to elucidate structures of activated FFAR1 bound to a G_q mimetic, which were induced either by the endogenous FFA ligand docosahexaenoic acid or γ-linolenic acid and the agonist drug TAK-875. Our data identify the orthosteric pocket for fatty acids and show how both endogenous hormones and synthetic agonists induce changes in helical packing along the outside of the receptor that propagate to exposure of the G-protein-coupling site. These structures show how FFAR1 functions without the highly conserved "DRY" and "NPXXY" motifs of class A GPCRs and also illustrate how the orthosteric site of a receptor can be bypassed by membrane-embedded drugs to confer full activation of G protein signaling.

Keywords: FFAR1; GPCR; cryoEM; fatty acid; insulin.

Fig. 1.

Overall structures of activated FFAR1-G protein complexes. (A, Top) cryo-EM reconstruction of FFAR1-mG_sqiN bound to DHA. Receptor density is gold, mG_sqiN is magenta, G_β1 is green, G_γ2 is yellow, and scFv16 is purple. Cryo-EM density from Relion was displayed in Chimera as colored surfaces (contoured at 4.9 sigma), where different colored zones correspond to the different polypeptides. (A, Bottom) the corresponding model is shown as a cartoon, with DHA agonist in green spheres. (B, Top) cryo-EM reconstruction of FFAR1-mG_sqiN with TAK-875 and γLA. Receptor density is blue, mG_sqiN is magenta, G_β1 is green, G_γ2 is yellow, and scFv16 is purple. Cryo-EM density from Relion was displayed in Chimera as colored surfaces (contoured at 5.9 sigma), where different colored zones correspond to the different polypeptides. (B, Bottom) the corresponding model is shown as a cartoon, with TAK-875 agonist in cyan spheres. (C, Top) cryo-EM reconstruction of FFAR1-mG_sqiN with TAK-875 and γLA in lipid nanodisc. Receptor density is purple, mG_sqiN is magenta, G_β1 is green, G_γ2 is yellow, and scFv16 is purple. Cryo-EM density from Relion was displayed in Chimera as colored surfaces (contoured at 6.1 sigma), where different colored zones correspond to the different polypeptides. (C, Bottom) the corresponding model is shown as a cartoon, with TAK-875 agonist in cyan spheres.

Fig. 2.

Orthosteric ligand binding to activated FFAR1. (A) Superposition of activated FFAR1 bound to DHA (gold cartoon, green sticks), activated FFAR1 bound to TAK-875 (blue cartoon, cyan sticks), and inactive FFAR1 bound to TAK-875 (gray cartoon, gray sticks, PDB: 4PHU). (B) Contact residues (gold sticks) within 4 Å of DHA (green sticks) in activated FFAR1. Polar contacts within 4 Å are shown as dashed orange lines. (C) Contact residues (blue sticks) within 4 Å of TAK-875 (cyan sticks) in activated FFAR1. Polar contacts within 4 Å are shown as dashed orange lines. (D) Stimulation of G_q by FFAR1 wild type (WT) and mutants in response to DHA (Left), γLA (Middle), and TAK-875 (Right). Each data point in the IP accumulation assay represents the average from three or more independent experiments, each performed in duplicate. Error bars denote ± SEM. Data were normalized to the WT E_max and fitted to the four-parameter model “log(agonist) vs. response” in GraphPad Prism 9. Statistical parameters are shown in SI Appendix, Table S2A.

Fig. 3.

Functional characterization of the allosteric site in activated FFAR1. (A) Position of orthosteric and allosteric sites mapped onto the structure of activated FFAR1 (gold cartoon, DHA complex). The predicted positions of orthosteric agonist MK-8666 (red sticks) and AgoPAM ligand AP8 (purple sticks) were determined by the superposition of the inactive FFAR1 structure (PDB 5TZY) with our activated DHA-bound FFAR1. (B) Stimulation of G_q by FFAR1 wild type (WT) and A102^3.48 mutants in response to DHA (Top) or γLA (Bottom). Each data point in the IP accumulation assay represents the average from three or more independent experiments, each performed in duplicate. Error bars denote ± SEM. Data were normalized to the WT E_max and fitted to the four-parameter model “log(agonist) vs. response” in GraphPad Prism 9. Statistical parameters are shown in SI Appendix, Table S2C. (C-D) Stimulation of G_q by FFAR1 in response to TAK-875 in the presence of increasing concentrations of DHA (C) or γLA (D). Each data point in the IP accumulation assay represents the average from three or more independent experiments, each performed in duplicate. Error bars denote ± SEM. Data were normalized to the WT (E_max defined as 100%) and fitted to the four-parameter model “log(agonist) vs. response” in GraphPad Prism 9. Statistical parameters are shown in SI Appendix, Table S2B.

Fig. 4.

Molecular dynamics (MD) simulation of activated FFAR1. (A) Plot of SD (σ) of perpendicular distances from all the C_α atoms of TM3 to the best-fit line of the C_α atoms, over time of the simulation. The red trace (Apo) corresponds to the simulation of the activated FFAR1 structure in the absence of the DHA ligand. The fast decline of the σ value starting with the activated structure (DHA complex) indicates that the extracellular half of TM3 becomes straighter during the first 75 nanoseconds of the MD simulation. The black trace (DHA) corresponds to the simulation of the activated FFAR1 structure starting with the DHA ligand intact. (B) Superposition of FFAR1 before (white cartoon) and after (cyan cartoon) the Apo MD simulation. TM3 at the end of the simulation is colored green to highlight the change in conformation of its extracellular half to become straighter in the absence of a ligand.

Fig. 5.

Activation of FFAR1 and interface with G protein. (A) FFAR1–mG_sqiN complex bound to DHA. FFAR1 is a gold cartoon, G_α is magenta, and DHA is in green spheres. The Inset shows an enlarged view of the interface between FFAR1 and α5-helix of G_αq, with FFAR1 residues as gold sticks and G_q residues as magenta sticks. Polar contacts <4 Å are highlighted as dashed orange lines. (B) Overlay of NPXXY motif between activated FFAR1 (DHA complex, residues shown as gold sticks) and inactive FFAR1 (PDB: 4PHU, residues shown as gray sticks). (C) Overlay of DRY motif between activated FFAR1 (DHA complex, residues shown as gold sticks) and inactive FFAR1 (PDB: 4PHU, residues shown as gray sticks). (D) Overlay of a toggle switch between activated FFAR1 (DHA complex, residues shown as gold sticks) and inactive FFAR1 (PDB: 4PHU, residues shown as gray sticks). (E) Overlay of PIF motif between activated FFAR1 (DHA complex, residues shown as gold sticks) and inactive FFAR1 (PDB: 4PHU, residues shown as gray sticks). (F) Superposition of receptors with G protein a5 helix in FFAR1-G_q (gold cartoon), β₂AR-G_s (slate blue cartoon, PDB:3SN6), and M1R-G₁₁ (cyan cartoon, PDB: 6OIJ).

Fig. 6.

Allosteric switch in FFAR1 and membrane-embedded AgoPAMs. (A, Left) Superposition of activated FFAR1 (DHA complex, gold cartoon and sticks) with inactive FFAR1 (PDB: 4PHU, gray sticks), showing the movement of helices (purple arrows). (A, Right Top) Change in packing of residues on TM4 and TM5 between inactive (gray cylinders and sticks) and activated FFAR1 (gold cylinders and sticks). (A, Right Bottom) Change in packing of TM domains at the intracellular surface between inactive (gray cylinders and sticks) and activated FFAR1 (gold cylinders and sticks). (B) Superposition of activated FFAR1 (DHA complex, gold cartoon and sticks) with AP8-bound inactive FFAR1 (PDB: 5TZY, purple cartoon and sticks). (C) Superposition of AP8-bound inactive FFAR1 (PDB: 5TZY, purple cartoon and sticks), compound 1–bound inactive FFAR1 (PDB: 5KW2, light purple cartoon and sticks), Compd-6FA-bound β₂AR (PDB: 6N48, green cartoon and sticks), and LY3154207-bound D1R (PDB: 7X2F, yellow cartoon and sticks). The inset shows the AgoPAM site bound to monoolein in TAK-875-bound FFAR1 (PDB: 4PHU, gray cartoon and orange sticks).