The molecular basis of ligand interaction at free fatty acid receptor 4 (FFA4/GPR120)

Abstract

The long-chain fatty acid receptor FFA4 (previously GPR120) is receiving substantial interest as a novel target for the treatment of metabolic and inflammatory disease. This study examines for the first time the detailed mode of binding of both long-chain fatty acid and synthetic agonist ligands at FFA4 by integrating molecular modeling, receptor mutagenesis, and ligand structure-activity relationship approaches in an iterative format. In doing so, residues required for binding of fatty acid and synthetic agonists to FFA4 have been identified. This has allowed for the refinement of a well validated model of the mode of ligand-FFA4 interaction that will be invaluable in the identification of novel ligands and the future development of this receptor as a therapeutic target. The model reliably predicted the effects of substituent variations on agonist potency, and it was also able to predict the qualitative effect of binding site mutations in the majority of cases.

Keywords: 7-Helix Receptor; Bioluminescence Resonance Energy Transfer (BRET); Diabetes; Fatty Acid; G Protein-coupled Receptor (GPCR); Homology Modeling.

FIGURE 1.

Structures and potencies of ligands used in this study at human FFA4 and FFA1. Potencies were determined using the β-arrestin-2 recruitment assay.

FIGURE 2.

Cell surface expression is correlated with β-arrestin-2 response efficacy for FFA4 mutants. Correlation plots of receptor-β-arrestin-2 BRET response efficacy versus measured cell surface expression are shown for aLA (A), TUG-891 (B), TUG-670 (C), and GW9508 (D). Mutants that gave no ligand-mediated response were excluded from these analyses. Correlation coefficients are also shown.

FIGURE 3.

GW9508 has a distinct mode of binding compared with aLA, TUG-891, and TUG-670. Ca²⁺ assays show the effect of F88A^2.53 and F311A^7.43 mutations on the potency of aLA (A), TUG-891 (B), TUG-670 (C), and GW9508 (D). These assays demonstrate that although F88A^2.53 shows reduced potency for all ligands except GW9508, F311A^7.43 in contrast displays reduced potency only for GW9508. The binding pose of GW9508 compared with aLA, TUG-891, and TUG-670 is shown in E. GW9508 (green) is positioned at a longer distance (3.3 Å) from Phe-88 than aLA (2.5 Å), TUG-891 (2.4 Å), and TUG-670 (2.7 Å) (all blue) when docked in this model, in agreement with the lack of effect for the F88A^2.53 mutation on this ligand.

FIGURE 4.

Orthosteric binding site of FFA4 in complex with aLA, TUG-891, TUG-670, and GW9508. The binding poses of aLA (A), TUG-891 (B), TUG-670 (C), and GW9508 (D) are shown with the side chains of all residues that significantly affect potency when mutated to Ala.

FIGURE 5.

Comparison of calculated binding energies and experimental potencies obtained for various ligands and mutant forms of FFA4. Relative effects of mutations on binding energies predicted from docking in mutant receptor models (ΔΔG (kcal/mol), red bars, scale on left side) and experimentally determined potency (pEC₅₀, green bars, scale on right side, inactive in blue) for aLA (A) TUG-891 (B), TUG-670 (C), and GW9508 (D).

FIGURE 6.

ortho-Biphenyl binding pocket of FFA4. The binding pocket formed by Phe-88^2.53, Thr-119^3.33, Gly-122^3.36, Trp-277^6.48, Thr-310^7.42, Asn-215^5.46, Val-212^5.43, Phe-211^5.42, Ile-280^6.51, and Ile-281^6.52. Residues are shown in orange, and TUG-891 is shown in green.

FIGURE 7.

Effect of Ala, Phe, and Val mutations at position Ile-281^6.52 of FFA4 on the potency of ortho-biphenyl compounds. Concentration-response curves for TUG-891 (A), TUG-670 (B), TUG-854 (C), and TUG-827 (D) at each of wild type (circles), I281A^6.52 (squares), I281F^6.52 (triangles), and I281V^6.52 (inverted triangle) FFA4 in the β-arrestin-2 BRET assay.

FIGURE 8.

Effects of the I281A^6.52 mutation on biphenyl ligands with and without the 4′-methyl substituent. The poses of TUG-891 (A) and TUG-827 (B) are minimally affected by the I281A^6.52 mutation (0.79 and 0.82 Å, respectively), whereas TUG-670 (C) and TUG-804 (D) shift further into the pocket (1.59 and 1.68 Å, respectively). Ala-281^6.52 shown as blue spheres overlapped by Ile-281^6.52 shown as transparent cyan spheres. The structures docked in the wild type FFA4 model are cyan, and structures docked in the I281A^6.52 mutant model are blue.

FIGURE 9.

Terminal 4′-methyl group of TUG-891 is in direct contact with Val-212. Compounds TUG-670 (left) and TUG-891 (right) docked in the wild type FFA4. Phe-211^5.42, Val-212^5.43, Ile-280^6.51 (not shown), and Ile-281^6.52 form a pocket around the terminal ring of the biphenyl system.