Characterization of the β-d-Glucopyranoside Binding Site of the Human Bitter Taste Receptor hTAS2R16

Taishi Sugawara,Keiko Abe,Katsuyoshi Masuda,Takuya Kobayashi,Takumi Misaka,Masaji Ishiguro,Tomiko Asakura,Yoshiro Ishimaru,Shinji Matsuo,Takanobu Sakurai,Keisuke Ito

doi:10.1074/jbc.m110.144444

Abstract

G-protein-coupled receptors mediate the senses of taste, smell, and vision in mammals. Humans recognize thousands of compounds as bitter, and this response is mediated by the hTAS2R family, which is one of the G-protein-coupled receptors composed of only 25 receptors. However, structural information on these receptors is limited. To address the molecular basis of bitter tastant discrimination by the hTAS2Rs, we performed ligand docking simulation and functional analysis using a series of point mutants of hTAS2R16 to identify its binding sites. The docking simulation predicted two candidate binding structures for a salicin-hTAS2R16 complex, and at least seven amino acid residues in transmembrane 3 (TM3), TM5, and TM6 were shown to be involved in ligand recognition. We also identified the probable salicin-hTAS2R16 binding mode using a mutated receptor experiment. This study characterizes the molecular interaction between hTAS2R16 and β-d-glucopyranoside and will also facilitate rational design of bitter blockers.

Highlights

There are five basic and distinct taste sensations: sweetness, bitterness, saltiness, sourness, and umami
The TAS1R family of taste receptors mediates two taste sensations; TAS1R2/TAS1R3 responds to sweetness [2, 3] and TAS1R1/TAS1R3 responds to umami taste [2, 4], whereas the TAS2R family responds to bitterness [5,6,7,8,9,10,11,12,13,14]
Humans recognize thousands of compounds as bitter, and response to bitterness is mediated by the hTAS2R family, which are G-protein-coupled receptors composed of only 25 receptors

Summary

EXPERIMENTAL PROCEDURES

Compounds—Salicin, arbutin, and phenyl-␤-D-glucopyranoside were selected as test ligands for hTAS2R16 (Fig. 1) [7]. If salicin binding is mediated as Through the above approach, two binding modes of sali- in model A, the response of these mutants to salicin would not cin in the ligand binding region were suggested as candidates change, because both Phe and Tyr residues have aromatic rings for the salicin-receptor complex structure Trp-94 and Gln-177, were targeted for point mutagenesis, and analysis of Trp-94 and Gln-177 mutants above, the binding their responses to salicin were examined using a cell-based mode for salicin in hTAS2R16 of model B is preferred over that assay for HEK293T cells cotransfected with the ssr3- of model A (Fig. 2B). In model B, Trp-94 seemed to be the Glu-86 mutants E86D and E86Q using arbutin and phenylinvolved in CH-␲ interactions and in hydrogen ␤-D-glucopyranoside as the ligands to verify whether or not hTAS2R16-salicin binding mode accords with model B

Predicted amino acid residues involved in salicin recognition

No response

DISCUSSION

Asakura and Keiko Abe