Conserved Residues Control the T1R3-Specific Allosteric Signaling Pathway of the Mammalian Sweet-Taste Receptor

Abstract

Mammalian sensory systems detect sweet taste through the activation of a single heteromeric T1R2/T1R3 receptor belonging to class C G-protein-coupled receptors. Allosteric ligands are known to interact within the transmembrane domain, yet a complete view of receptor activation remains elusive. By combining site-directed mutagenesis with computational modeling, we investigate the structure and dynamics of the allosteric binding pocket of the T1R3 sweet-taste receptor in its apo form, and in the presence of an allosteric ligand, cyclamate. A novel positively charged residue at the extracellular loop 2 is shown to interact with the ligand. Molecular dynamics simulations capture significant differences in the behavior of a network of conserved residues with and without cyclamate, although they do not directly interact with the allosteric ligand. Structural models show that they adopt alternate conformations, associated with a conformational change in the transmembrane region. Site-directed mutagenesis confirms that these residues are unequivocally involved in the receptor function and the allosteric signaling mechanism of the sweet-taste receptor. Similar to a large portion of the transmembrane domain, they are highly conserved among mammals, suggesting an activation mechanism that is evolutionarily conserved. This work provides a structural basis for describing the dynamics of the receptor, and for the rational design of new sweet-taste modulators.

Keywords: allosteric binding site; class C GPCR; cyclamate; mammalian; sweet-taste receptor; taste modulator.

Figure 1.

Schematic of the sweet-taste receptor structure. VFD, CRD, TMD. This figure is reproduced in color in the online version of the issue.

Figure 2.

(a) Schematic representation of the ligand–receptor interactions. Charged (R725^ecl2 and R790^7.28), polar (Q636^3.32, Q637^3.33, S640^3.36, H641^3.37, H721^ecl2, S726^ecl2, S729^5.39, and Q794^7.32), and hydrophobic residues (F624^2.56, F730^5.40, F778^6.53, L782^6.57, and P791^7.29) are represented as purple, blue and green spheres (dark and light grey in the print version), respectively. (b) Representative structure of the cyclamate-bound T1R3 receptor obtained from molecular dynamics simulations. The binding mode is consistent with the best docking solution and recapitulates available experimental data. Residues of the allosteric binding pocket and those involved in the transmission switch are respectively shown as blue and green (light grey in the print version) sticks. (c) Activity of wild-type sweet-taste receptor and single-point mutants of functional residues upon application of 10-mM d-tryptophan (■) and cyclamate (●); each experiment was repeated 3 times. (Statistical significance: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.) (d-f) d-tryptophan (■) and cyclamate (●) dose–response curves obtained for wild-type sweet-taste receptor (d) and single-point mutants of residue R725^ecl2 (e), N737^5.47, Y771^6.46, and W775^6.50 (f); each experiment was repeated twice.

Figure 3

Structural analysis of the apo (a) and the agonist-bound (b) forms of the T1R3 receptor. Representative structures display the conformations of residues N737^5.47, Y771^6.46, and W775^6.50. Red arrows suggest a putative activation mechanism. (c) Distribution of the inter-residue N737^5.47-Y771^6.46 and N737^5.47-W775^6.50 distances (1 and 2). Distribution of the Y771^6.46-W775^6.50 side-chain center-of-mass distances (3). Distances have been calculated between the O^δ1, O^η, and N^ε1 atoms of N737^5.47, Y771^6.46, and W775^6.50, respectively. Y771^6.46 center of mass has been calculated considering the aromatic ring including C^ε1, C^ε2, C^δ1, C^δ2, C^γ, and C^ζ atoms. W775^6.50 center of mass has been calculated considering the pyrrole ring including N^ε1, C^ε2, C^δ1, C^δ2, C^γ atoms. See Supplementary Figure 4 for time series plots.

Figure 4.

Meta-analysis of T1R3 TMD site-directed mutagenesis data. Experimental data have been summarized from Jiang et al. (2005b) and Winnig et al. (2007), represented by triangle and square symbols. New site-directed mutagenesis experiments reported in the present study are indicated by filled circles. Receptor responses indicate the effect of mutations on the receptor activity, expressed as a percentage compared to the wild-type receptor, to the allosteric (cyclamate in x axis) and orthosteric (d-trp and aspartame in y axis) ligands. Cyclamate-specific interacting residues are in blue. Residues that control the signaling pathway or abolish the receptor response are colored in green and red, respectively. Residues that have no or weak-specific effect on receptor response are in black. All data are summarized in Supplementary Table 3.