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Identification of key neoculin residues responsible for the binding and activation of the sweet taste receptor.

Koizumi T, Terada T, Nakajima K, Kojima M, Koshiba S, Matsumura Y, Kaneda K, Asakura T, Shimizu-Ibuka A, Abe K, Misaka T - Sci Rep (2015)

Bottom Line: We found that the mutations of Arg48, Tyr65, Val72 and Phe94 of NCL basic subunit increased or decreased both the antagonist and agonist activities.The mutations had only a slight effect on the pH-dependent functional change.From these results, we concluded that NCL interacts with hT1R2-hT1R3 through a pH-independent affinity interface including the four residues and a pH-dependent activation interface including the histidine residues.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.

ABSTRACT
Neoculin (NCL) is a heterodimeric protein isolated from the edible fruit of Curculigo latifolia. It exerts a taste-modifying activity by converting sourness to sweetness. We previously demonstrated that NCL changes its action on the human sweet receptor hT1R2-hT1R3 from antagonism to agonism as the pH changes from neutral to acidic values, and that the histidine residues of NCL molecule play critical roles in this pH-dependent functional change. Here, we comprehensively screened key amino acid residues of NCL using nuclear magnetic resonance (NMR) spectroscopy and alanine scanning mutagenesis. We found that the mutations of Arg48, Tyr65, Val72 and Phe94 of NCL basic subunit increased or decreased both the antagonist and agonist activities. The mutations had only a slight effect on the pH-dependent functional change. These residues should determine the affinity of NCL for the receptor regardless of pH. Their locations were separated from the histidine residues responsible for the pH-dependent functional change in the tertiary structure. From these results, we concluded that NCL interacts with hT1R2-hT1R3 through a pH-independent affinity interface including the four residues and a pH-dependent activation interface including the histidine residues. Thus, the receptor activation is induced by local structural changes in the pH-dependent interface.

No MeSH data available.


Related in: MedlinePlus

Evaluation of the sweetness of the NCL double mutants.(A) Separate locations of the residues responsible for the pH-dependency (green) or the agonist and antagonist potencies (yellow) are indicated on the diagram showing the three-dimensional backbone of NCL. The backbones of NAS and NBS are coloured in pale red and pale blue, respectively. The right diagram depicts the lateral side of NBS. (B) Cell-based analysis of the sweetness of the NCL double mutants. The responses of cells expressing hT1R2-hT1R3 and G15Gi3 were examined after the application of NCL double mutants carrying one substitution from each group of residues. All four double mutants showed equivalent activation of the cells under neutral (pH 7.4) and weakly acidic (pH 6.3) conditions. Additionally, their activation potencies were increased or decreased compared to the NBS H11A single-point mutant depending on the nature of the other mutation. The number of responsive cells was normalised relative to the maximum response to aspartame (6.7 mM) at pH 7.4. Error bars represent the mean ± SE (n = 4). *P < 0.05, ***P < 0.001 vs. NBS H11A single-point mutant (one-way ANOVA followed by Tukey’s post hoc test).
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f4: Evaluation of the sweetness of the NCL double mutants.(A) Separate locations of the residues responsible for the pH-dependency (green) or the agonist and antagonist potencies (yellow) are indicated on the diagram showing the three-dimensional backbone of NCL. The backbones of NAS and NBS are coloured in pale red and pale blue, respectively. The right diagram depicts the lateral side of NBS. (B) Cell-based analysis of the sweetness of the NCL double mutants. The responses of cells expressing hT1R2-hT1R3 and G15Gi3 were examined after the application of NCL double mutants carrying one substitution from each group of residues. All four double mutants showed equivalent activation of the cells under neutral (pH 7.4) and weakly acidic (pH 6.3) conditions. Additionally, their activation potencies were increased or decreased compared to the NBS H11A single-point mutant depending on the nature of the other mutation. The number of responsive cells was normalised relative to the maximum response to aspartame (6.7 mM) at pH 7.4. Error bars represent the mean ± SE (n = 4). *P < 0.05, ***P < 0.001 vs. NBS H11A single-point mutant (one-way ANOVA followed by Tukey’s post hoc test).

Mentions: Figure 4A shows the distribution of the residues that contribute to NCL’s activity. The residues important for pH-dependency (NAS Tyr21 and His36 and NBS His11 and His14) are located separately from the residues important for both the agonist and antagonist potencies (NBS Arg48, Tyr65, Val72, and Phe94) in the tertiary structure. This result prompted us to evaluate whether the residues of these two groups function individually or cooperatively. For this purpose, four double mutants carrying one substitution from each group were produced. NBS His11 was substituted with Ala in all of the double mutants, whereas one of the Arg48, Tyr65, Val72, and Phe94 residues of NBS was substituted with Ala in each double mutant. Each double mutant protein was applied to cells expressing hT1R2-hT1R3 together with G15Gi3 under neutral (pH 7.4) or weakly acidic (pH 6.3) conditions. As shown in Fig. 4B, all four double mutants showed equivalent activation of the receptor at neutral and weakly acidic pH due to the loss of the pH-dependency regulated by NBS His11. Additionally, the activation level was increased compared to the NBS H11A mutant for the H11A/R48A and H11A/V72A double mutants and was decreased for the H11A/Y65A and H11A/F94A double mutants. Importantly, these tendencies are the same as those observed in the experiment using the single-point mutants (Fig. 3E). Therefore, the effects of the two groups on NCL’s activity are additive, suggesting that the residues of the two groups function individually.


Identification of key neoculin residues responsible for the binding and activation of the sweet taste receptor.

Koizumi T, Terada T, Nakajima K, Kojima M, Koshiba S, Matsumura Y, Kaneda K, Asakura T, Shimizu-Ibuka A, Abe K, Misaka T - Sci Rep (2015)

Evaluation of the sweetness of the NCL double mutants.(A) Separate locations of the residues responsible for the pH-dependency (green) or the agonist and antagonist potencies (yellow) are indicated on the diagram showing the three-dimensional backbone of NCL. The backbones of NAS and NBS are coloured in pale red and pale blue, respectively. The right diagram depicts the lateral side of NBS. (B) Cell-based analysis of the sweetness of the NCL double mutants. The responses of cells expressing hT1R2-hT1R3 and G15Gi3 were examined after the application of NCL double mutants carrying one substitution from each group of residues. All four double mutants showed equivalent activation of the cells under neutral (pH 7.4) and weakly acidic (pH 6.3) conditions. Additionally, their activation potencies were increased or decreased compared to the NBS H11A single-point mutant depending on the nature of the other mutation. The number of responsive cells was normalised relative to the maximum response to aspartame (6.7 mM) at pH 7.4. Error bars represent the mean ± SE (n = 4). *P < 0.05, ***P < 0.001 vs. NBS H11A single-point mutant (one-way ANOVA followed by Tukey’s post hoc test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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f4: Evaluation of the sweetness of the NCL double mutants.(A) Separate locations of the residues responsible for the pH-dependency (green) or the agonist and antagonist potencies (yellow) are indicated on the diagram showing the three-dimensional backbone of NCL. The backbones of NAS and NBS are coloured in pale red and pale blue, respectively. The right diagram depicts the lateral side of NBS. (B) Cell-based analysis of the sweetness of the NCL double mutants. The responses of cells expressing hT1R2-hT1R3 and G15Gi3 were examined after the application of NCL double mutants carrying one substitution from each group of residues. All four double mutants showed equivalent activation of the cells under neutral (pH 7.4) and weakly acidic (pH 6.3) conditions. Additionally, their activation potencies were increased or decreased compared to the NBS H11A single-point mutant depending on the nature of the other mutation. The number of responsive cells was normalised relative to the maximum response to aspartame (6.7 mM) at pH 7.4. Error bars represent the mean ± SE (n = 4). *P < 0.05, ***P < 0.001 vs. NBS H11A single-point mutant (one-way ANOVA followed by Tukey’s post hoc test).
Mentions: Figure 4A shows the distribution of the residues that contribute to NCL’s activity. The residues important for pH-dependency (NAS Tyr21 and His36 and NBS His11 and His14) are located separately from the residues important for both the agonist and antagonist potencies (NBS Arg48, Tyr65, Val72, and Phe94) in the tertiary structure. This result prompted us to evaluate whether the residues of these two groups function individually or cooperatively. For this purpose, four double mutants carrying one substitution from each group were produced. NBS His11 was substituted with Ala in all of the double mutants, whereas one of the Arg48, Tyr65, Val72, and Phe94 residues of NBS was substituted with Ala in each double mutant. Each double mutant protein was applied to cells expressing hT1R2-hT1R3 together with G15Gi3 under neutral (pH 7.4) or weakly acidic (pH 6.3) conditions. As shown in Fig. 4B, all four double mutants showed equivalent activation of the receptor at neutral and weakly acidic pH due to the loss of the pH-dependency regulated by NBS His11. Additionally, the activation level was increased compared to the NBS H11A mutant for the H11A/R48A and H11A/V72A double mutants and was decreased for the H11A/Y65A and H11A/F94A double mutants. Importantly, these tendencies are the same as those observed in the experiment using the single-point mutants (Fig. 3E). Therefore, the effects of the two groups on NCL’s activity are additive, suggesting that the residues of the two groups function individually.

Bottom Line: We found that the mutations of Arg48, Tyr65, Val72 and Phe94 of NCL basic subunit increased or decreased both the antagonist and agonist activities.The mutations had only a slight effect on the pH-dependent functional change.From these results, we concluded that NCL interacts with hT1R2-hT1R3 through a pH-independent affinity interface including the four residues and a pH-dependent activation interface including the histidine residues.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.

ABSTRACT
Neoculin (NCL) is a heterodimeric protein isolated from the edible fruit of Curculigo latifolia. It exerts a taste-modifying activity by converting sourness to sweetness. We previously demonstrated that NCL changes its action on the human sweet receptor hT1R2-hT1R3 from antagonism to agonism as the pH changes from neutral to acidic values, and that the histidine residues of NCL molecule play critical roles in this pH-dependent functional change. Here, we comprehensively screened key amino acid residues of NCL using nuclear magnetic resonance (NMR) spectroscopy and alanine scanning mutagenesis. We found that the mutations of Arg48, Tyr65, Val72 and Phe94 of NCL basic subunit increased or decreased both the antagonist and agonist activities. The mutations had only a slight effect on the pH-dependent functional change. These residues should determine the affinity of NCL for the receptor regardless of pH. Their locations were separated from the histidine residues responsible for the pH-dependent functional change in the tertiary structure. From these results, we concluded that NCL interacts with hT1R2-hT1R3 through a pH-independent affinity interface including the four residues and a pH-dependent activation interface including the histidine residues. Thus, the receptor activation is induced by local structural changes in the pH-dependent interface.

No MeSH data available.


Related in: MedlinePlus