Free fatty acid receptors: structural models and elucidation of ligand binding interactions

Abstract

Background: The free fatty acid receptors (FFAs), including FFA1 (orphan name: GPR40), FFA2 (GPR43) and FFA3 (GPR41) are G protein-coupled receptors (GPCRs) involved in energy and metabolic homeostasis. Understanding the structural basis of ligand binding at FFAs is an essential step toward designing potent and selective small molecule modulators.

Results: We analyse earlier homology models of FFAs in light of the newly published FFA1 crystal structure co-crystallized with TAK-875, an ago-allosteric ligand, focusing on the architecture of the extracellular binding cavity and agonist-receptor interactions. The previous low-resolution homology models of FFAs were helpful in highlighting the location of the ligand binding site and the key residues for ligand anchoring. However, homology models were not accurate in establishing the nature of all ligand-receptor contacts and the precise ligand-binding mode. From analysis of structural models and mutagenesis, it appears that the position of helices 3, 4 and 5 is crucial in ligand docking. The FFA1-based homology models of FFA2 and FFA3 were constructed and used to compare the FFA subtypes. From docking studies we propose an alternative binding mode for orthosteric agonists at FFA1 and FFA2, involving the interhelical space between helices 4 and 5. This binding mode can explain mutagenesis results for residues at positions 4.56 and 5.42. The novel FFAs structural models highlight higher aromaticity of the FFA2 binding cavity and higher hydrophilicity of the FFA3 binding cavity. The role of the residues at the second extracellular loop used in mutagenesis is reanalysed. The third positively-charged residue in the binding cavity of FFAs, located in helix 2, is identified and predicted to coordinate allosteric modulators.

Conclusions: The novel structural models of FFAs provide information on specific modes of ligand binding at FFA subtypes and new suggestions for mutagenesis and ligand modification, guiding the development of novel orthosteric and allosteric chemical probes to validate the importance of FFAs in metabolic and inflammatory conditions. Using our FFA homology modelling experience, a strategy to model a GPCR, which is phylogenetically distant from GPCRs with the available crystal structures, is discussed.

Fig. 1

Agonists of the free fatty acid 1–3 receptors. The potency of compounds was taken from ref. 5, 9 and 13

Fig. 2

The superimposition of the FFA1 crystal structure and homology models base on the backbone of the helices in the extracellular side. The crystal structure, rhodopsin, β₂ adrenergic and PAR₁-based homology models are in yellow, cyan, pink and grey colour, respectively. Residues predicted to be important for ligand coordination based on mutagenesis and residue K62^2.60, representing the possible anchoring point for allosteric ligands are shown in stick-like representation. The green arrows indicate the large movement of helices 3, 4 and 5 in the FFA1 crystal structure compared to the homology models

Fig. 3

The ligand binding mode at the FFA1 crystal structure and homology models. a: the binding model of TAK-875, the ago-allosteric ligand in the FFA1 crystal structure, the ligand is pointed between helices 3 and 4 (mode 1 in the text). b: The binding mode of GW9508, the high potency agonist in the previous rhodopsin-based homology model of FFA1. The model was obtained from ref 12. c: The binding mode of TAK-875 in the PAR₁-based model of FFA1. Hydrogen bonds are in black

Fig. 4

The superimposition of the FFA1 crystal structure and the homology model of the FFA1 wild type bound to TAK-875. The FFA1 wild type model was constructed from the FFA1 crystal structure using Prime [43]. TAK-875 was docked to the wild type model using Glide [20]. Phenylalanine mutation at position 3.34 is shown in pink

Fig. 5

Two proposed binding modes for the GW9508 agonist in FFA1. a: mode 1, similar to the binding of TAK-875 in the FFA1 crystal structure. b: mode 2, the alternative pose derived from flexible docking. Hydrogen bonds, π- π and π-cation interactions are in black and blue, respectively. The binding site residues, E145 and E172 of the second extracellular loop predicted to form salt bridges in the earlier models and K62^2.60 predicted here to form the allosteric binding site are shown

Fig. 6

The ligand binding site of the free fatty acid 1–3 receptors a: the binding site in the FFA1 crystal structure; b and c: the binding site in the FFA1-based homology model of FFA2 and FFA3, respectively. The side chain of the binding site residues in FFAs is shown in blue, pink and green, respectively. H-bonding, π- π and π-cation interactions are shown in black, blue and green dotted lines, respectively

Fig. 7

Ligand binding in FFA2 and FFA3. a: the binding mode of tiglic acid at FFA2. b: the binding mode of 1-MCPC in FFA3. c: the binding mode of 1 in FFA2. FFA2-3 homology models are based on the FFA1 crystal structure. H-bonding and π- π interactions are shown in black and blue dotted lines, respectively