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

Abstract

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 α-helical (AH) and Ras-like domains of Gα subunit from a precoupled TAS2R5-G protein state to the fully activated state. A moderate agonist opens the AH-Ras cleft from 22 Å to 31 Å 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 Gα 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 Gα 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.

Keywords: FLOWER; G protein-coupled receptor; airway smooth muscle; bitter taste receptor; metadynamics.

Fig. 1.

D9 cells transfected to express TAS2R5 were plated in 96-well plates, loaded with Fluo-4AM, and exposed to the indicated concentrations of the agonists. The ionomycin (IONO) concentration was 2 μM. (A–C) are representative experiments showing the time-dependent increases in [Ca²⁺]_i from the indicated agonists. (D–F) are of mean data (normalized to the IONO response) fit to a sigmoid curve (n = 7 to 10). Table 1 for mean values of the EC₅₀, E_Max, and S.T coefficients.

Fig. 2.

The recorded signal from three different agonists binding to the TAS2R5 receptor. (A) The recorded binding signal in wavelength resonant shift over time for T5-14 binding to the TAS2R5 receptor, (B) a representative resonance dip. The Q-factor is calculated from the full-width half maximum (FWHM). (C–E) The wavelength shift response of each indicated agonist from representative experiments. Table 1 shows the mean K_D values from multiple experiments.

Fig. 3.

(A) The equilibrated structure of the preactivated TAS2R5-G_i protein complex. (B–D) Binding sites of TAS2R5 with T5-1, T5-8, and T5-14, respectively (Side view). (E) Superimposed structures of three different agonists in the binding site (Top view).

Fig. 4.

Snapshot structures of metaD simulations for (A) apo and (B) T5-8-bound TAS2R5 representing closed and open conformations of Gα subunit, respectively. MetaD energetics associated with opening the Gα subunit as a function of the distance between the AH domain (the center of mass of Cαs for residues 62 to 182) from the Ras-like domain (the center of mass of Cαs for residues 42 to 58, and 272 to 280, and 326 to 335) for the TAS2R5-G_i protein with (C) T5-1 showing ΔG = −4.8 kcal/mol, (D) T5-8 showing ΔG = −11.1 kcal/mol, (E) T5-14 showing ΔG = −1.4 kcal/mol, and (F) apo showing ΔG = +6.9 kcal/mol.

Fig. 5.

Transitions from the precoupled state to the activated state induced by the binding of T5-1, T5-8, and T5-14, alongside the opening of Gα subunit. Agonist potencies correlate more strongly with the energetics of Gα subunit opening than with their binding affinities.