1. Introduction
The human sweet taste receptor is a heterodimeric complex of the proteins T1R2 and T1R3. The complex is a member of the G-protein-coupled receptor class (GPCR); which share a common design of a seven transmembrane heptahelical domain with an extracellular N-terminus and intracellular C-terminus. The downstream signaling pathway of the receptor is thought to be controlled by G-proteins that stimulate the synthesis of intracellular second messengers such as cyclic AMP, inositol phosphate and Ca
2+ ions. The sweet GPCR has been further grouped as a class C receptor, of which there are 4 other families: class A rhodopsin family, class B secretin family, class D adhesion family and class E frizzles/smoothened family. The class C GPCR share a common structure of a large amino-terminal domain (ATD), which serves as the principle ligand-binding domain, followed by a short cysteine-rich domain (CRD) tied to the transmembrane domain (TMD) and intracellular C-terminal domain [
1,
2].
The sweet-taste receptor has been shown to bind a large ensemble of molecules such as sugars, artificial sweeteners, sweet-tasting proteins and some D-amino acids that mediate the sweet taste response (
Figure 1A). Regions of the complex that bind specific ligands include the ATD of human (h) T1R2: non-caloric sweeteners aspartame, neotame, sucralose and monellin, a sweet-tasting protein; the ATD of T1R3: cyclamate, neohesperidin dihydrochalcone and lactisole; and the ATD and CRD of hT1R3: sweet-tasting proteins brazzein and neoculin [
3,
4,
5,
6,
7]. T1R2 and T1R3 subunits have been shown to bind natural sugars glucose and sucrose with distinct affinities even though the individual contributions of each subunit to the interaction are unknown.
One of the major difficulties in studying the molecular details of the function of this complex has been the lack of a reliable method for producing large quantities of purified proteins using recombinant technology. The mouse (m) versions of T1R2 and T1R3 ATDs have been successfully produced, but only in small quantities and as fusion proteins [
8]. Recently, hT1R3 ATD was purified and characterized [
9]. Here, we describe multi-modal screening methodologies that are required for the complex heterodimeric sweet taste receptor. First, we will describe a method for producing highly purified protein constructs for both human and mouse fusion small ubiquitin-like modifier (SUMO)-T1R2 ATD protein and the protease cut T1R2 ATD protein [
10]. Gel filtration chromatography demonstrated that the T1R2 ATD exists in a dimeric configuration. Second, we will describe several complementary methods for the study of the constructs and their interaction with small ligands that elicit a sweet-taste response in tests conducted in vivo with human taste panels [
11] and in vitro by heterologous calcium assays with HEK211 cells overexpressing the sweet taste receptor [
12]. Using circular dichroism spectroscopy, we show that there is a decrease in the overall α-helical content of the construct upon binding to sweet ligands. In addition, there appears to be an overall decrease in thermal stability in the tertiary structure of the SUMO-hT1R2 ATD fusion protein upon binding of neotame, a small sweet-taste inducing dipeptide ligand. Saturation transfer difference spectra confirmed that molecules eliciting a CD response also gave positive difference spectra indicating binding of ligands to the ATD domain. Furthermore, we show that combination of intrinsic fluorescence spectroscopy, circular dichroism spectroscopy and saturation transfer difference spectroscopy can be useful in evaluating detailed molecular changes at the receptor level while monitoring ligand binding. These complementary methods provide important tools for studying allosteric effects of one sweet-taste responding molecule over another, as described by the heterologous calcium assay for sweet-taste response [
12]. In this work, we demonstrate that we can study the highly purified sweet-taste protein and its binding of target molecules using these biophysical methods.
3. Discussion
Currently, sweet receptor extracellular domains are difficult to study due to their large size and unfavorable properties. The sweet receptor is heterodimeric consisting of T1R2/T1R3 subunits. The receptor contains large extracellular domains. These large domains mediate the interaction of ATDs with small natural and synthetic sweeteners. T1R2 ATD contains proposed binding pockets for dipeptides (e.g., neotame, aspartame), proteins (brazzein, monellin), and small natural and artificial sweeteners (
Figure 1A). In order to understand the molecular mechanism by which the sweet taste response is generated, we have used a set of methodologies to measure binding events that correlate with previous sweet taste assays. Earlier work [
8] described the ATD region of T1R3 as a soluble, folded and functional protein. We extend the work now to include the equivalent portion of human T1R2 and compared it with mouse T1R2, which is known to have species-specific binding properties. We have used the recombinant protein that contains the ATD region of human T1R2 as a model system to target small molecule ligands. Furthermore, we demonstrate that the ATD region of human or mouse T1R2 is capable of dimerization on its own, as monitored by gel filtration chromatography, and is able to bind to sweet ligands with micromolar binding constants (
Table 1). These results are in agreement with previous studies of the full heterodimeric receptor (T1R2/T1R3) [
3,
4,
12].
The combination of biophysical tools (CD, STD-NMR and DSC) provide a roadmap for studying the binding mechanism of small molecules, prior to downstream events that lead to a sweet taste response. In addition, we show that MSG, a ligand specific to the umami receptor (T1R1), does not bind to ATDs of T1R2. Similarly, our tests show that the SUMO protein tag is not involved in the binding process and could be used as a negative control (data not shown). CD spectroscopy shows a loss of secondary structure in the human ATD upon its binding to neotame, while DSC shows destabilization of the protein-ligand complex. It is likely that this thermal destabilization is correlated with the loss of α-helical content upon binding that was observed by CD. CD measurements in the near-UV region of the spectrum are clearly valuable to obtain both qualitative and quantitative information. As such, we used this sensitive tool to calculate dissociation constants for a number of sweet ligands in the presence of ATD h-T1R2 protein (
Table 1). Furthermore, the use of the STD-NMR technique allowed direct observation of binding interaction of neotame with the extracellular domain of T1R2 subunit. Although STD-NMR could be used for determining apparent dissociation constants these measurements should be taken with care and require optimization of several experimental conditions to avoid overestimating
KD values [
13]. To overcome limitations in each particular biophysical measurement, we combined these complementary molecular tools to access ligand binding interaction modes. In addition, our approaches are complementary to the existing calcium imaging assay in cells that require a fully functional heterodimeric receptor (T1R2/T1R3) to report on the receptor response to sweeteners. In addition, our platform provides in depth analysis of new sweeteners either alone or in competition and/or inhibition studies to fully understand the structure/functional role of ATD region of T1R2 that transpires in the initial binding event prior to sweet taste response.
Recently, Dong and colleagues [
14] have used fullerenol as a model for the sweet-taste receptor to investigate the binding affinities of structural enantiomers of sweet-taste ligands. Their basic findings demonstrated a correlation between sweet intensity and binding energy. Our methodologies would be well suited to confirm these results using the actual protein-binding interface. In addition, these results would give a more detailed description of the events that lead to either a productive or destructive binding event.
In addition, other laboratories [
12] have used taste receptor molecules to study the binding of various small sweet molecules. In particular, Masuda et al. studied the cellular responses to sweet taste stimulus in transiently transfected cells and cellular responses to sweet taste stimulus by monitoring calcium flux. The calcium-imaging assay is a downstream event that yields very little information on the molecular actions that involve a sweet-taste response in the receptor itself. Our model system for studying these interactions, in contrast, can lead to uncovering the seminal details that occur in a productive sweet taste response.
In conclusion, we suggest the application of these complementary methods to a wide range of research in other GPCR receptors. Just to name a few, we can use our expression and purification system on the equivalent ATD regions of T1R3 and T1R1, and use site-directed mutagenesis to elucidate important amino acids in these binding events. We can also study a much wider range of sweet tasting ligands, as well as those that are inhibitors (such as phenoxy herbicides) or enhancers of sweet taste response.
4. Materials and Methods
4.1. Production and Purification of Mouse and Human T1R2 ATD Constructs
The ATD region of the protein was cloned into a 8xHis tag vector with either a TEV cleavage site or in a SUMO vector [
10]. The human constructs ranged from residues Asn24-Met494 for human (ATD-hT1R2) in the SUMO fusion, or Ser25-Thr489 in the 8xHis-Tag. The mouse 8xHis-Tev-ATD-mT1R2 construct ranged from residues Gly2-Pro466. DNA coding for the amino terminal ligand binding domain of the sweet taste receptor from ATDs was expressed as inclusion bodies in
Escherichia coli BL21-CodonPlus(DE3)-RIPL cells grown at 37 °C in 1 L of LB medium. The purification follows a procedure that has been previously described, with some modifications [
9,
10]. The inclusion bodies were solubilized in 6 M guanidinium chloride and refolded by dialysis against a buffer containing 50 mM Tris-acetate (pH 8.0), 50 mM KCl, 2 mM Zwittergent 3-14 and 2 mM DTT. The amino terminal domain (ATD) was purified to homogeneity on a Superdex 200 prep grade FPLC column and analyzed by 12% SDS-PAGE. The protein concentration was determined by the Bradford method using bovine serum albumin as the standard [
15]. The yield from the 1 L of culture media was 5–10 mg of final protein.
4.2. Saturation Transfer Difference Spectroscopy (STD-NMR)
The ligand binding activity of ATD-T1R2 for all ligands was confirmed with a NMR-STD binding assay as previously described [
11,
16]. Aliquots of the pure labeled SUMO-T1R2 protein were incubated with desired titrating ligands at 0.5–20 fold molar excess and concentrated to a final concentration of ~0.05 mM in 10 mM phosphate buffer (pH 7.4) containing 150 mM NaCl, 2.7 mM KCl, 5 mM DTT, 5x protease inhibitor (Roche) and 0.05% NaN
3. Monosodium glutamate (MSG, non-sweet molecule) was used as a negative control. NMR data were collected on a VNMRS spectrometer (Varian, Palo Alto, CA, USA) operating at 800 MHz and equipped with a cryogenic probe. STD-NMR data collection and analysis was carried out as previously described [
13].
4.3. Circular Dichroism Spectroscopy (CD)
CD spectra were recorded at 25 °C on an Model 202SF circular dichroism spectrometer (Aviv, Lakewood, NY, USA) equipped with a Peltier temperature control. Samples were added to a 0.01 cm path length quartz cuvette with a concentration of the ATD (from human and mouse) of about 0.3 mg/mL (4.5 μM) in 10 mM Tris-HCl, 150 mM NaCl, 10% glycerol, pH 7.4. Data was collected every 1 nm with an averaging time of 5 s. The spectral bandwidth was 1 nm. Spectra were corrected for buffer and ligands contributions and converted to mean ellipticity in deg cm
2·dmol
−1. The content of α-helix was computed using the deconvolution program from Chen et al. [
16]. Titrating concentrations for ligands were from zero (no ligand present) to maximum 3 mM (above saturating concentrations) in the presence of fixed concentration (4.5 μM) of the ATD protein. The
KD was calculated by analyzing CD data at a fixed wavelength versus ligand concentration using a nonlinear regression method (see
Table 1) [
17]. MSG was used as a negative control.
4.4. Intrinsic Fluorescence Spectroscopy (Fl)
Samples were prepared in the same way as for the CD experiments. Experiments were performed at 25 °C on Carey Eclipse instrument (Agilent, Santa Clara, CA, USA). Spectra were subtracted from the buffer ± ligand backgrounds for presentation. The curves at 340 nm were the best fits of the data obtained by a regression method using Microsoft Excel software (Microsoft, Redmont, WA, USA).
4.5. Differential Scanning Calorimetry (DSC)
DSC was performed using a VP-DSC microcalorimeter (Microcal, Malvern, United Kingdom). Samples of His-T1R2 ATD at about 20 mM were dialyzed against 4 × 1 L of 10 mM Tris-HCl, 150 mM NaCl, 10% glycerol, pH 7.4 in the absence or presence of 0.2 mM ligand for 16 h at 4° C prior to use. Buffer without protein was hermetically sealed in the reference and sample compartments and repeated thermograms were generated from 10–95 °C and at 1 °C/min. After confirming that the repeated buffer/buffer thermoprofiles were identical, the protein sample was exchanged in the sample compartment and scanned against the reference buffer. Data was analyzed using the Microcal software package. The thermograms were background subtracted, normalized for concentration to ∆Cp (mcal·deg−1) and baselines established on the pre- and post-transitional data. The Tm (°C) and ∆H (kcal·mol−1) were determined from the corrected curves using non-2-state transitional curve fitting.