Agonist activation to open the Gα subunit of the GPCR-G protein precoupled complex defines functional agonist activation of TAS2R5

G protein- coupled receptors (GPCRs) regulate multiple cellular responses and represent highly successful therapeutic targets. The mechanisms by which agonists activate the G protein are unclear for many GPCR families, including the bitter taste receptors (TAS2Rs). We ascertained TAS2R5 properties by live cell- based functional assays, direct binding affinity measurements using optical resonators, and atomistic molecular dynamics simulations. We focus on three agonists that exhibit a wide range of signal transduction in cells despite comparable ligand-receptor binding energies derived from direct experiment and computation. Metadynamics simulations revealed that the critical barrier to activation is ligand- induced opening of the G protein between the alpha- helical (AH) and Ras- like domains of G alpha subunit from a precoupled TAS2R5- G protein state to the fully activated state. A moderate agonist opens the AH- Ras cleft from 22 & Aring; to 31 & Aring; with an energy gain of -4.8 kcal mol-1, making GDP water- exposed for signaling. A high- potency agonist had an energy gain of -11.1 kcal mol-1. The low- potency agonist is also exothermic for G alpha opening, but with an energy gain of only -1.4 kcal mol-1. This demonstrates that TAS2R5 agonist- bound functional potencies are derived from energy gains in the transition from a precoupled complex at the level of G alpha opening. Our experimental and computational study provides insights into the activation mechanism of signal transduction that provide a basis for rational design of new drugs.