Dynamics of Ligand Binding Sites and Chloride Penetration in a Bitter Taste GPCR.

Taste perception strongly influences food choice, drug compliance, and dietary behavior. Bitter taste receptors belong to a subfamily of Family A GPCRs, while having unique features and deviations from conserved motifs. Recent cryo-EM structures revealed that the human bitter taste receptor TAS2R14 contains not only the canonical extracellular binding site, but also a novel intracellular site, raising questions about their interplay. Here, we performed all-atom molecular dynamics simulations of TAS2R14 in an explicit membrane to probe different receptor states: both cholesterol (extracellular) and aristolochic acid (intracellular) bound, only cholesterol bound, only aristolochic acid bound, and the apo receptor, with and without the G protein. Ligand occupancy at either site reshaped the volume, hydration, and conformational plasticity of both pockets and modulated the relative proximity of transmembrane helices 3 and 6. In simulations lacking the intracellular agonist but containing cholesterol, we observed intracellular entry of chloride ions and their long-lived interaction with the TAS2R-conserved R55 (BW position 2.50), revealing a charge-inverted interaction relative to the D2.50-sodium interaction described in many class A GPCRs. Apo simulations without G protein showed shorter-lived chloride visits to the same region. Together, these findings point to the intracellular pocket as a promising target for future mechanistic and ligand-design studies on bitter taste GPCRs.