Abstract
Humans' bitter taste perception is mediated by the hTAS2R subfamily of the G protein-coupled membrane receptors (GPCRs). Structural information on these receptors is currently limited. Here we identify residues involved in the binding of phenylthiocarbamide (PTC) and in receptor activation in one of the most widely studied hTAS2Rs (hTAS2R38) by means of structural bioinformatics and molecular docking. The predictions are validated by site-directed mutagenesis experiments that involve specific residues located in the putative binding site and trans-membrane (TM) helices 6 and 7 putatively involved in receptor activation. Based on our measurements, we suggest that (i) residue N103 participates actively in PTC binding, in line with previous computational studies. (ii) W99, M100 and S259 contribute to define the size and shape of the binding cavity. (iii) W99 and M100, along with F255 and V296, play a key role for receptor activation, providing insights on bitter taste receptor activation not emerging from the previously reported computational models.
Highlights
Humans, like other mammals, have evolutionary been prevented from the ingestion of a large variety of poisonous and toxic substances by their aversion for bitter tasting food [1,2,3,4]
Screening hTAS2R38 for residues involved in PTC binding We constructed an ensemble of a few hundred of structural models of the receptor based on comparative homology modeling
This was achieved by aligning the hTAS2R38 sequence with those of all G proteincoupled receptors (GPCRs) whose structure has been solved (Figure S1)
Summary
Like other mammals, have evolutionary been prevented from the ingestion of a large variety of poisonous and toxic substances by their aversion for bitter tasting food [1,2,3,4]. First principle [16] and homology modeling approaches based on bovine rhodopsin [17] have been used to predict the structure of the widely studied bitter taste receptor hTAS2R38 [21,22] Both works call upon further computational refinement and/or experimental validations. The degree of sequence conservation across the GPCRs superfamily, and the human bitter taste receptor subfamily (TAS2Rs) in particular, is very low In this scenario, experimental validation improves greatly homology-based models [23,24]. This procedure is likely not to be sufficient to identify residues in the binding site as ligand pockets vary largely in position and orientation across this family [14] Addressing this issue is aided by predicting the threedimensional structure of the receptor, based on the former alignment and recent structural information on GPCRs along with massive virtual docking calculations. The proposed receptor positions are scrutinized by sitedirected mutagenesis experiments and measurements of receptor activation by recording intracellular calcium levels following agonist administration
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