Structural basis for the ligand recognition and signaling of free fatty acid receptors

Abstract

Free fatty acid receptors 1 to 4 (FFA1 to FFA4) are class A G protein-coupled receptors (GPCRs). FFA1 to FFA3 share substantial sequence similarity, whereas FFA4 is unrelated. However, FFA1 and FFA4 are activated by long-chain fatty acids, while FFA2 and FFA3 respond to short-chain fatty acids generated by intestinal microbiota. FFA1, FFA2, and FFA4 are potential drug targets for metabolic and inflammatory conditions. Here, we determined the active structures of FFA1 and FFA4 bound to docosahexaenoic acid, FFA4 bound to the synthetic agonist TUG-891, and butyrate-bound FFA2, each complexed with an engineered heterotrimeric G_q protein (miniG_q), by cryo-electron microscopy. Together with computational simulations and mutagenesis studies, we elucidated the similarities and differences in the binding modes of fatty acid ligands to their respective GPCRs. Our findings unveiled distinct mechanisms of receptor activation and G protein coupling. We anticipate that these outcomes will facilitate structure-based drug development and underpin future research on this group of GPCRs.

Fig. 1.. Overall structures of FFA1, FFA2, and FFA4 signaling complexes.

(A) Overall structures of DHA-FFA4 (slate), DHA-FFA1 (green), TUG-891-FFA4 (dark yellow), and butyrate-FFA2 (blue), each in complex with miniG_q, together with the chemical structures of the bound ligands. miniG_αq, G_β, and G_γ subunits are colored salmon, cyan, and light blue, respectively. ScFv16 is colored gray. The LCFA EPA is also shown for comparison to DHA (see the main text for discussion). (B) Comparison of the above structures as seen from the intracellular face.

Fig. 2.. Ligand binding in FFA4.

(A to D) Details of the interactions of DHA (orange) and TUG-891 (purple) with FFA4. (A) illustrates the general positions of the two ligands, while (B) highlights the closed nature of the occupied ligand-binding pockets. Details of key residues of the binding pockets are highlighted for DHA (C) and TUG-891 [(D), left]. TUG-1197 docked into the FFA4 structure [(D), right] highlights the important role of T119 and the similarity of the binding mode of TUG-1197 and TUG-891 at the bottom of the pocket. (E) Various point mutants of FFA4 generated and assessed for the ability of each of TUG-891, TUG-1197, and DHA to promote interactions with arrestin-3. See fig. S7 for quantitation. In concert with the large-scale mutagenesis studies reported previously (42), this provides a comprehensive analysis of the orthosteric binding pocket of FFA4. (F) Continuous electron density observed between W198 and E204. The cryo-EM map is contoured at the level of 0.13. (G) Negative charge potential of the FFA4 binding pocket with DHA. (H) Additional length of DHA compared to EPA and the position of the DHA carboxylate above and beyond E204.

Fig. 3.. Potential DHA binding sites in FFA1.

(A) DHA binding in site 1. The partial cryo-EM density map of DHA colored light blue in the left panel is contoured at the level of 0.07. C1, C8, and C22 atoms of DHA are labeled. The occupancy of DHA C9-C22 was assigned as zero due to a lack of density. The details of interactions between DHA and FFA1 in site 1 are shown in the right panel. DHA is colored brown. (B) Putative DHA binding in site 2. The strong cryo-EM density map in this site is contoured at the level of 0.12. The modeled DHA molecule is colored gray. Polar interactions are shown as black dashed lines. FFA1 is colored green. (C) DHA concentration-response curves at the wild-type receptor (WT) and Y114A mutant measured by arrestin-3 interaction assays and relative expression of WT (black), Y114A (blue), and Y44A (red) FFA1 measured by eYFP fluorescence shown on top. Data are means ± SEM for n = 3 or more.

Fig. 4.. Recognition of butyrate by FFA2.

(A) Structural alignment of FFA2-butyrate and FFA1-DHA. The carboxylate of butyrate occupies the equivalent position to the carboxylate of DHA. (B) Details of interactions between butyrate and FFA2. (C) Overall shape of the butyrate binding pocket. (D) Differences in location of TM3 and TM4 in FFA1 and FFA2. In all panels, FFA1 and FFA2 are colored green and blue, respectively, while DHA and butyrate are colored brown and yellow, respectively. Polar interactions are shown as black dashed lines.

Fig. 5.. Agonist recognition of FFA4 and FFA2 probed by MD simulations.

(A) FFA4-DHA, (B) FFA4–TUG-891, and (C) FFA2-butyrate complexes. A representative frame is shown with key residues forming contacts with DHA (orange) with DHA carbon-carbon double bonds numbered 1 to 6, and TUG-891 (purple) and butyrate (yellow) in stick representation. The size and color of the residues correspond to the average strength of van der Waals and electrostatic interactions with the agonist, respectively. Water clusters observed in the MD simulations are shown in the cyan surface-like representation. The superscripts in the amino acid labels denote the Ballesteros–Weinstein generic GPCR residue numbering.

Fig. 6.. Activation of FFAs.

(A) Superimposition of the active DHA-bound FFA4 structure (slate) to the Alphafold-predicted inactive FFA4 structure FFA4-AF (light gray) viewed from the intracellular (left) and the extracellular (right) sides. (B) Residues involved in the receptor activation at the core region of FFA4. (C) Superimposition of the active butyrate-bound FFA2 structure (blue) to the Alphafold-predicted FFA2 structure FFA2-AF (dark gray) viewed from the intracellular (left) and the extracellular (right) sides. (D) Superimposition of the active DHA-bound FFA1 structure (green) to the Alphafold-predicted FFA1 structure FFA1-AF (light blue) viewed from the intracellular side. Red solid and dashed arrows represent conformational changes of TMs and individual residues, respectively, from the Alphafold-predicted structures to the active agonist-bound structures of FFA1, FFA2, and FFA4.

Fig. 7.. Differences in the coupling of miniG_q to FFAs.

(A) Alignment of the structures of FFA1, FFA2, and FFA4 coupled with miniG_q based on the receptors. (B) Differences in the interactions between miniG_αq and ICL2 of FFA1, FFA2, and FFA4. (C) Differences in the interactions between miniG_αq and ICL3 of FFA1, FFA2, and FFA4. (D) Superimposition of the AlphaFold-predicted structure of FFA4^Long to the structure of DHA-bound FFA4 coupled with miniG_q. MiniG_αq, G_β, and G_γ subunits are colored salmon, cyan, and light blue, respectively. The colors of receptors and ligands are indicated in each panel.