Interaction of Food-Derived Bitter Peptides with hTAS2R4: Recognition and Activation Mechanisms Revealed by Docking and Mutagenesis

Bitter peptides (BPs) have a wide range of applications in the food and pharmaceutical industries. This study identified four food-derived BPs (TPLVDR, VLY, LEEL, and FYL) as novel agonists of the human bitter taste receptor hTAS2R4, using a rapid cell-based screening approach. Activation of hTAS2R4 by these peptides inhibited downstream cAMP signaling, with effective concentration values of 191.99, 222.96, 297.17, and 63.96 μM, respectively. Molecular docking and sequence alignment across 25 bitter taste receptors highlighted eight potential binding sites. Subsequent site-directed mutagenesis validated F62 2.57 , F69 2.64 , V85 3.29 , S184 5.461 , Y250 6.56 , and L266 7.38 as key binding sites for hTAS2R4 activation by BPs. These findings provided crucial evidence for the molecular mechanism of peptide recognition and signal transduction by hTAS2R4 activation.