Activation of G protein-coupled receptors entails cysteine modulation of agonist binding

The increase of the affinity of agonists with an increase in pH and experiments using thiol-specific reagents indicate that G protein-coupled receptors contain an ionizable cysteine residue at the ligand binding site. Since treatment of receptors with reducing agents produces functional activation and potentiates agonist stimulation, it is likely that this free sulfhydryl modulates receptor activation. We have derived a two-state acid-base model for cysteine modulation of ligand binding which leads to a description of ligand efficacy. We have shown that pH-dependent binding of agonists is closely correlated with measurements of ligand efficacy at the 5-HT2A receptor. In general, efficacy is determined by the preference of a ligand for the base of the receptor. Efficacy may also be described in thermodynamic terms as the coupling free energy involving a ligand and the acid and base states of the receptor. Molecular modeling of the third transmembrane domain of the 5-HT2A receptor, which contains a conserved cysteine, shows that efficacy is determined by the difference between the electrostatic interaction energies of a ligand with the acid and base forms of the receptor model. The difference in interaction energy between the two forms of cysteine makes the largest contribution to this electrostatic interaction energy difference. Therefore, the cysteine makes the largest contribution to ligand efficacy. Using this approach, we can distinquish between the efficacies of agonists with varying molecular structures and account for the differences between the properties of agonists and antagonists

Insights into the Binding of Phenyltiocarbamide (PTC) Agonist to Its Target Human TAS2R38 Bitter Receptor

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

Molecular dynamics of a biophysical model for b 2-adrenergic

and G protein-coupled receptor activation