Direct NMR detection of the binding of functional ligands to the human sweet receptor, a heterodimeric family 3 GPCR

Abstract

We present a robust method for monitoring the binding of ligands to the heterodimeric (T1R2+T1R3) human sweet receptor (a family 3 GPCR receptor). The approach utilizes saturation transfer difference (STD) NMR spectroscopy with receptor proteins expressed on the surface of human epithelial kidney cells. The preparation investigated by NMR can contain either live cells or membranes isolated from these cells containing the receptor. We have used this approach to confirm the noncompetitive binding of alitame and cyclamate to the receptor and to determine that greatly reduced receptor binding affinity compared to wild-type brazzein explains the lack of sweetness of brazzein mutant A16C17. This approach opens new avenues for research on the mechanism of action of the sweet receptor and for the design of new noncalorigenic sweeteners.

Figure 1

Scheme illustrating the use of STD NMR to monitor interactions between a membrane receptor and ligand. A ¹H NMR pulse applied selectively to the receptor is rapidly transferred by spin diffusion throughout the receptor. This saturation is transferred to a bound ligand, which, upon its release from the receptor, adds to the pool of saturated ligand present in excess over the receptor. The build-up of saturation in the ligand pool is governed by a number of factors, including the on and off rates for ligand binding, the rotational correlation time for the complex, and the relaxation rates of ligand signals.

Figure 2

Evidence that the heterodimeric sweet taste receptor is expressed and displayed on the of human embryonic kidney (HEK293) cells. Monoclonal mouse–Flag for hT1R2-flag with Alexa488-conjugated goat–mouse secondary antibodies (upper right) and polyclonal rabbit–T1R3 antibody with Alexa594-conjugated goat–rabbit secondary antibodies (lower left) were used to assess the stable heterologous expression of sweet taste receptor in HEK293 cells in the merge image (lower right) as compared to total cell density in transmission (upper left) to account for those cells not expressing either T1R2 or T1R3. Results showed <1% non-expressing cells; ~1% hT1R3-only expressing cells; and ~5% hT1R2-only expressing cells. The parental cell line showed no signal with either antibody (data not shown).

Figure 3

Comparison of the interaction between (A) wild-type and (B) non-sweet brazzein mutant A16C17 with the wild-type sweet receptor (hT1R2+hT1R3) in membranes isolated from HEK+r cells as detected by one-dimensional ¹H-¹⁵N HSQC saturation transfer double difference spectroscopy (STDD). Membranes (75–100 μg) were resuspended in 150 μL perdeuterated PBS (phosphate buffered saline: 10 mM Na-phosphate buffer, 137 mM NaCl, and 2.7 mM KCl, pH 7.4). [U-¹⁵N]-Brazzein or A16C17 (3–5 mg) was added to the membrane preparation prior to data collection. NMR data were collected on a Varian 800 MHz spectrometer equipped with a cryogenic probe. Top trace: experiment (negative control). Middle trace: STD spectra of membrane preparations containing the receptor minus STD spectra of membrane preparations without receptor (STDD). Bottom trace: 1D ¹⁵N-selected ¹H NMR spectra of each brazzein variant.