Key amino acid residues involved in multi-point binding interactions between brazzein, a sweet protein, and the T1R2-T1R3 human sweet receptor

Abstract

The sweet protein brazzein [recombinant protein with sequence identical with the native protein lacking the N-terminal pyroglutamate (the numbering system used has Asp2 as the N-terminal residue)] activates the human sweet receptor, a heterodimeric G-protein-coupled receptor composed of subunits Taste type 1 Receptor 2 (T1R2) and Taste type 1 Receptor 3 (T1R3). In order to elucidate the key amino acid(s) responsible for this interaction, we mutated residues in brazzein and each of the two subunits of the receptor. The effects of brazzein mutations were assayed by a human taste panel and by an in vitro assay involving receptor subunits expressed recombinantly in human embryonic kidney cells; the effects of the receptor mutations were assayed by in vitro assay. We mutated surface residues of brazzein at three putative interaction sites: site 1 (Loop43), site 2 (N- and C-termini and adjacent Glu36, Loop33), and site 3 (Loop9-19). Basic residues in site 1 and acidic residues in site 2 were essential for positive responses from each assay. Mutation of Y39A (site 1) greatly reduced positive responses. A bulky side chain at position 54 (site 2), rather than a side chain with hydrogen-bonding potential, was required for positive responses, as was the presence of the native disulfide bond in Loop9-19 (site 3). Results from mutagenesis and chimeras of the receptor indicated that brazzein interacts with both T1R2 and T1R3 and that the Venus flytrap module of T1R2 is important for brazzein agonism. With one exception, all mutations of receptor residues at putative interaction sites predicted by wedge models failed to yield the expected decrease in brazzein response. The exception, hT1R2 (human T1R2 subunit of the sweet receptor):R217A/hT1R3 (human T1R3 subunit of the sweet receptor), which contained a substitution in lobe 2 at the interface between the two subunits, exhibited a small selective decrease in brazzein activity. However, because the mutation was found to increase the positive cooperativity of binding by multiple ligands proposed to bind both T1R subunits (brazzein, monellin, and sucralose) but not those that bind to a single subunit (neotame and cyclamate), we suggest that this site is involved in subunit-subunit interaction rather than in direct brazzein binding. Results from this study support a multi-point interaction between brazzein and the sweet receptor by some mechanism other than the proposed wedge models.

Figure 1

A. Backbone structure of brazzein showing the three “sweet sites” determined previously . The numbering system used is that for the wild-type variant that does not contain pGlu1 and has Asp2 as the N-terminal residue. B. Schematic representation of the GPCR sweet sensor T1R2-T1R3 heterodimer: VFTM, the extracellular Venus fly trap module; CRD, the extracellular cysteine-rich domain; TMD, the 7 transmembrane α-helices; C-term, the C-terminal intracellular tail. Arrows represent binding locations proposed for several ligands: the sweeteners alitame, D-trp (VFTM of T1R2) and cyclamate (TMD of T1R3), the anti-sweet agent lactisol (TMD of T1R3), whereas brazzein binding locations are to VFTM of T1R2 and CRD of T1R3.

Figure 2

Wedge models of interactions between the sweet receptor and sweet proteins. Homology-modeled structures of the Venus fly trap modules of T1R2 and T1R3, with the Cysteine-rich domains (CRD). T1R2 is shown in blue; T1R3 is shown in yellow. (Top panels) A1. T1R3-closed/T1R2-open, B1. T1R2-closed/T1R3-open. Brazzein (grey) has been predicted to fit in the cavity of the mouth of the VFTM of either T1R2 (A) or T1R3 (B), and to contact interface proximal residues from the other subunit. The binding surface of the receptor to brazzein predicted by the wedge models ; ; ; are represented as a mesh envelope with highlighted surfaces. An expanded view of this region is represented in the bottom panels. (Bottom panels) A2. Ball-and-stick representation of the residues that might be involved in brazzein binding when fitted in the open-VFTM cleft of T1R2: T1R3-E172; T1R2:E145,Y215. B2. Ball-and-stick of the residues that might be involved in brazzein binding when fitted in the open-VFTM cleft of T1R3: T1R2:E170,R172,D173,R217; T1R3:E148,H145,Y218. Stick representation of other potentially interacting residues are also displayed in A2 and B2.

Figure 3

Profile of the responses of the heterologously expressed human sweet receptor to a panel of sweeteners by the calcium mobilization assay. Human T1R2 and human T1R3 heterodimer sweet taste receptors were expressed in HEK293 cells along with the promiscuous G-protein chimera G16-gus44. Activity of the human sweet receptor in response to sweeteners was quantified by calcium imaging: all sweeteners, except sucrose and brazzein, were at saturating concentrations. HBSS buffer (used as dilution buffer for sweeteners) provides a negative control for sweet responses. Calcium mobilization is quantified as the peak of fluorescence response over baseline (ΔF/F) and normalized to the response of neotame. Data are mean ± SE of a single representative experiment.

Figure 4

A. Surface representation (left side “front” view; right side “back” view) of wild type brazzein showing the positions of mutations found to affect sweetness. Mutations that abolished sweetness are shown in dark blue; those that profoundly decreased sweetness relative to wild type are in medium blue; those that slightly decreased sweetness are in light blue; those that enhanced sweetness are in red. B. Location of the three “sweet sites”: Site 1 (Loop43), Site 2 (N- and C-termini, E36), and Site 3 (Loop9–19). The three sites form a non-continuous surface that spans about 18–20 Å ; .

Figure 5

A. Psychophysical analysis (taste panel) of wild type brazzein (WT) and a series of brazzein mutants. The study used brazzein protein concentrations of 100 μg/ml in comparison to a 2% solution of sucrose. Results from 10 subjects were averaged. The Y-axis indicates the sweetness score for wild type brazzein and its mutants in comparison with sucrose. Error bars represent mean ± SE. B. Activity of wild type brazzein and its mutants assayed by heterologous expression of the human sweet receptor (T1R2+T1R3) assayed by calcium mobilization in the presence of a reporter G-protein (G16-gus44). The calcium assay was carried out at four concentrations of wild type or mutant brazzeins: 100, 30, 10, and 3 μg/ml. The maximal response of the receptors in each experiment was quantified using a saturation concentration of the agonist neotame (50μM), and data were normalized to 100 % of this value. Values represent mean ± S.E of three independent experiments.

Figure 5

A. Psychophysical analysis (taste panel) of wild type brazzein (WT) and a series of brazzein mutants. The study used brazzein protein concentrations of 100 μg/ml in comparison to a 2% solution of sucrose. Results from 10 subjects were averaged. The Y-axis indicates the sweetness score for wild type brazzein and its mutants in comparison with sucrose. Error bars represent mean ± SE. B. Activity of wild type brazzein and its mutants assayed by heterologous expression of the human sweet receptor (T1R2+T1R3) assayed by calcium mobilization in the presence of a reporter G-protein (G16-gus44). The calcium assay was carried out at four concentrations of wild type or mutant brazzeins: 100, 30, 10, and 3 μg/ml. The maximal response of the receptors in each experiment was quantified using a saturation concentration of the agonist neotame (50μM), and data were normalized to 100 % of this value. Values represent mean ± S.E of three independent experiments.

Figure 6

Overlay of heteronuclear ¹⁵N-¹H HSQC spectra (fingerprint amide region) of wild type brazzein (black) and three “non-sweet” brazzein variants: L18_A19insRI ns (green), C16A/Q17C (red), C16A/C37A (blue). Psychophysical testing demonstrated that mutant L18_A19insRI, which contains two residues RI inserted between L18 and A19 of wild type brazzein, is tasteless. NMR chemical shift mapping of differences between this mutant and brazzein indicated that the residues affected by the insertion are localized to the mutated loop and to the region of the single α-helix.

Figure 7

The human T1R2 extracellular domain Venus-FlyTrap Module determines sensitivity to brazzein. Human (hT1R2+hT1R3), mouse (mT1R2+mT1R3) and chimeric mouse/human sweet receptors (as indicated by m/h prefix) and brazzein-permissible mouse T1R3 mutant T542A/P545F (mAF-R3) were activated by brazzein, cyclamate, neotame and sucralose and assayed for calcium responses. Chimeric receptors of T1R2 contained the extracellular portion (VFTM and CRD as for hm1-R2, or VFTM alone as for hm2-R2) of human receptor, and the CRD and/or only the transmembrane domain and C-terminal from mouse; as shown in the black/grey schematics receptor. Peak calcium signal is expressed as ΔF/F in %, normalized to maximal response to sucralose (5mM= ~25 EC₅₀). Data in the figure are from a representative experiment, means ± SE, done in quadruplicate. The star * represents P<0.001 difference of mT1R2 in comparison to the sweetener profile of hT1R2, when paired with mAF-T1R3.

Figure 8

Receptor mutants designed to test putative interaction sites in wedge models. WT and mutant receptor responses to two non-saturating concentrations of brazzein (50 and 250 μg/ml), sub-saturating concentrations of neotame (5 μM) and cyclamate (10 mM) and saturating concentration of sucralose (5 mM) were assayed by calcium mobilization. Peak calcium signal is expressed as ΔF/F in %, normalized to control (WT with sucralose). Values are means ± SE from at least three independent experiments. The star “*” represents P<0.001 difference of mutant receptors in comparison to WT sweetener profile. (Tables). Each “+” represents an increment of % of calcium response. “−”:≤ 10%. “+”:10><25. “++”:25><50. “+++”:50><75. “++++”:75><100. “+++++”:≥100. A and B. Neotame is a VFTM-T1R2 binding ligand and cyclamate is a TM-T1R3 binding ligand. Mutants T1R2-S144A^a , and T1R3-F730L^b , have been shown to specifically affect neotame and cyclamate response respectively. C and D. Mutants were chosen on the basis of the putative binding site(s) for brazzein shown in Figure 2. C. Residues that might be involved in brazzein binding when fitted in the open-VFTM cleft of T1R2: T1R3-E172; T1R2:E145,Y215. D. Residues that might be involved in brazzein binding when fitted in the open-VFTM cleft of T1R3: T1R2:E170,R172,D173,R217; T1R3:E148,H145,Y218.

Figure 9. The effect of receptor mutant T1R2:R217A on responses to the panel of sweetners

A. Dose-response analysis of neotame-activated (NTM) and brazzein-activated (braz) human receptor T1R2 + T1R3 and T1R2:R217A + T1R3. Peak calcium signal is expressed as ΔF/F, normalized to saturation. Data are merged from several experiments; data-points are means ± SD. Stars mark the positions of 50 and 250μg brazzein in order to facilitate comparisons to the responses of R217A in Sup. Fig. 1. B. EC₅₀ values and slope values (n_HILL) of the dose-responses curves to neotame, cyclamate, sucralose, brazzein and monellin for WT and T1R2:R217A mutant. Values are means ± SE from at least three independent experiments.