Bidirectional, iterative approach to the structural delineation of the functional "chemoprint" in GPR40 for agonist recognition

Abstract

GPR40, free fatty acid receptor 1 (FFAR1), is a member of the GPCR superfamily and a possible target for the treatment of type 2 diabetes. In this work, we conducted a bidirectional iterative investigation, including computational modeling and site-directed mutagenesis, aimed at delineating amino acid residues forming the functional "chemoprint" of GPR40 for agonist recognition. The computational and experimental studies revolved around the recognition of the potent synthetic agonist GW9508. Our experimentally supported model suggested that H137(4.56), R183(5.39), N244(6.55), and R258(7.35) are directly involved in interactions with the ligand. We have proposed a polarized NH-pi interaction between H137(4.56) and GW9508 as one of the contributing forces leading to the high potency of GW9508. The modeling approach presented in this work provides a general strategy for the exploration of receptor-ligand interactions in G-protein coupled receptors beginning prior to acquisition of experimental data.

Figure 1

Nucleotide and lipid receptor cluster. The names of sequences are presented according to the IUPHAR nomenclature. When IUPHAR name is not available, the gene name is shown. Synonyms are shown after the slash. The natural ligands are indicated in italics.

Figure 2

Representative conformations for each of the three clusters of GPR40 conformers. One hundred receptor conformers were divided into 12 groups based on the RMSD of specific residues (see text), which were subsequently clumped into three clusters on the basis of solvent accessible surface. The backbone of the receptor is represented as a yellow ribbon. The surfaces of the cavities are colored according to H-bonding properties (red H-bond donors, blue H-bond acceptors).

Figure 3

Docking poses for GW9508 generated using FLEXE, which combinatorially joined 12 protein conformations (see text). The carbon atoms are colored in cyan for pose1, in magenta for pose 2, and in green for pose 3.

Figure 4

Combined molecular interaction fields of carboxyl (red), hydrophobic (white), and aromatic (green) probes with 12 protein conformations. Only low interaction energy fields are shown.

Figure 5

Effect of mutations on the potency of agonist GW9508. The increase in intracellular calcium upon receptor activation was measured and expressed relative to the wild-type receptor as mean%±SEM (see Experimental Section). The mutations are indicated on each graph. Dashed lines show the response of the wild-type receptor performed in parallel with the mutant. The EC₅₀ of GW9508 in the wild-type receptor was 223 nM (log EC₅₀= −6.65 ± 0.028, n=18), with a range from 159 to 1,092 nM. The decrease in potency of GW9508 following different mutations were: >100 fold, R183A; >100 fold, R258A; >100 fold, R258K; 16 fold, N244A; 5.8 fold, H86F; 14 fold, H86A; 28 fold, H137F; >100 fold, H137A; and 1.1 fold, V237F. Data shown are averages of three or more experiment.

Figure 6

Arrangement of amino-aromatic interactions. A- Relative orientation of GW9508 obtained from docking study; B- the simplified system used for QM study; C – Result of QM calculation

Figure 7

The experimentally-supported binding site of GW9508 in GPR40 (on the left) and the simplified scheme of protein-ligand interactions (on the right).

Scheme 1

Agonists for GPR40