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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

NMR-spectroscopy-based screening of the residues most likely to undergo conformational changes.(A) Close-up views of the superposition of NCL 1H-15N HSQC spectra recorded during the pH-titration experiment. See Supplementary Fig. S2 for all regions. pH of each spectrum is indicated by the colour scale. (B) Classification of all NCL residues that showed significant chemical shift changes between pH 3 and 7. All the residues are classified as two groups: the residues that showed chemical shift changes primarily between pH 3 and 5 are classified as “group A”, whereas those that showed chemical shift changes primarily between pH 5 and 7 are classified as “group B”. The residues classified as “group A” are coloured in red, whereas those classified as “group B” are coloured in blue. The residues classified as neither group A nor group B are coloured in grey. (C) Mapping of the residues classified as “group B” on the protein surface of the crystal structure of NCL at pH 7.4 (PDB ID: 2D04). All of these residues (coloured in blue) were located near His residues (coloured in green). NBS Phe94 (coloured in cyan) was specially selected as the target of the subsequent alanine-scanning mutagenesis because it exhibited a significant chemical shift change in the pH range of 5–7, although it was classified as group A. The overall structure of NCL is shown in two orientations separated by 180°. The protein surfaces of NAS and NBS are coloured in pale red and pale blue, respectively. (D) Mapping of the residues whose signals were missing in the NMR spectra on the diagram showing the three-dimensional backbone of NCL (coloured in purple). The cysteine residues forming disulphide bonds are shown as ball-and-stick models. Some β-strands are labelled according to our previous paper (Shimizu-Ibuka et al.,25). The backbone of NAS and NBS are coloured in pale red and pale blue, respectively.
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f2: NMR-spectroscopy-based screening of the residues most likely to undergo conformational changes.(A) Close-up views of the superposition of NCL 1H-15N HSQC spectra recorded during the pH-titration experiment. See Supplementary Fig. S2 for all regions. pH of each spectrum is indicated by the colour scale. (B) Classification of all NCL residues that showed significant chemical shift changes between pH 3 and 7. All the residues are classified as two groups: the residues that showed chemical shift changes primarily between pH 3 and 5 are classified as “group A”, whereas those that showed chemical shift changes primarily between pH 5 and 7 are classified as “group B”. The residues classified as “group A” are coloured in red, whereas those classified as “group B” are coloured in blue. The residues classified as neither group A nor group B are coloured in grey. (C) Mapping of the residues classified as “group B” on the protein surface of the crystal structure of NCL at pH 7.4 (PDB ID: 2D04). All of these residues (coloured in blue) were located near His residues (coloured in green). NBS Phe94 (coloured in cyan) was specially selected as the target of the subsequent alanine-scanning mutagenesis because it exhibited a significant chemical shift change in the pH range of 5–7, although it was classified as group A. The overall structure of NCL is shown in two orientations separated by 180°. The protein surfaces of NAS and NBS are coloured in pale red and pale blue, respectively. (D) Mapping of the residues whose signals were missing in the NMR spectra on the diagram showing the three-dimensional backbone of NCL (coloured in purple). The cysteine residues forming disulphide bonds are shown as ball-and-stick models. Some β-strands are labelled according to our previous paper (Shimizu-Ibuka et al.,25). The backbone of NAS and NBS are coloured in pale red and pale blue, respectively.

Mentions: Next, we measured 1H-15N HSQC NMR spectra at pH 5, 6, and 7 (Fig. 2A and Supplementary Fig. S2A-B). Backbone 1HN and 15N resonances of these spectra were assigned by successively extrapolating the trend of the chemical shift changes to a higher pH. We observed 36 and 33 residues of NAS and NBS, respectively, that exhibited significant differences of more than 0.05 ppm in the 1H chemical shift or 0.5 ppm in the 15N chemical shift between the spectra at pH 3 and 7 (Supplementary Fig. S2C-F). Plotting the normalised chemical shift changes against pH demonstrated that the residues could be classified into two groups (Fig. 2A,B): in group A, most of the chemical shift changes occurred between pH 3 and 5, whereas in group B, most of the chemical shift changes occurred between pH 5 and 7. Because the change from an antagonist to an agonist of NCL occurs between neutral and weakly acidic pH, the residues of group B are more closely related to this function. Because all of these residues are located near His residues in the tertiary structure of NCL (Fig. 2C), the conformational change likely occur upon protonation of the His residues. Moreover, although Phe94 of NBS was classified as group A, it exhibited a significant chemical shift change in the pH range of 5–7. This result suggests that Phe94 is also related to this function.


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)

NMR-spectroscopy-based screening of the residues most likely to undergo conformational changes.(A) Close-up views of the superposition of NCL 1H-15N HSQC spectra recorded during the pH-titration experiment. See Supplementary Fig. S2 for all regions. pH of each spectrum is indicated by the colour scale. (B) Classification of all NCL residues that showed significant chemical shift changes between pH 3 and 7. All the residues are classified as two groups: the residues that showed chemical shift changes primarily between pH 3 and 5 are classified as “group A”, whereas those that showed chemical shift changes primarily between pH 5 and 7 are classified as “group B”. The residues classified as “group A” are coloured in red, whereas those classified as “group B” are coloured in blue. The residues classified as neither group A nor group B are coloured in grey. (C) Mapping of the residues classified as “group B” on the protein surface of the crystal structure of NCL at pH 7.4 (PDB ID: 2D04). All of these residues (coloured in blue) were located near His residues (coloured in green). NBS Phe94 (coloured in cyan) was specially selected as the target of the subsequent alanine-scanning mutagenesis because it exhibited a significant chemical shift change in the pH range of 5–7, although it was classified as group A. The overall structure of NCL is shown in two orientations separated by 180°. The protein surfaces of NAS and NBS are coloured in pale red and pale blue, respectively. (D) Mapping of the residues whose signals were missing in the NMR spectra on the diagram showing the three-dimensional backbone of NCL (coloured in purple). The cysteine residues forming disulphide bonds are shown as ball-and-stick models. Some β-strands are labelled according to our previous paper (Shimizu-Ibuka et al.,25). The backbone of NAS and NBS are coloured in pale red and pale blue, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4542694&req=5

f2: NMR-spectroscopy-based screening of the residues most likely to undergo conformational changes.(A) Close-up views of the superposition of NCL 1H-15N HSQC spectra recorded during the pH-titration experiment. See Supplementary Fig. S2 for all regions. pH of each spectrum is indicated by the colour scale. (B) Classification of all NCL residues that showed significant chemical shift changes between pH 3 and 7. All the residues are classified as two groups: the residues that showed chemical shift changes primarily between pH 3 and 5 are classified as “group A”, whereas those that showed chemical shift changes primarily between pH 5 and 7 are classified as “group B”. The residues classified as “group A” are coloured in red, whereas those classified as “group B” are coloured in blue. The residues classified as neither group A nor group B are coloured in grey. (C) Mapping of the residues classified as “group B” on the protein surface of the crystal structure of NCL at pH 7.4 (PDB ID: 2D04). All of these residues (coloured in blue) were located near His residues (coloured in green). NBS Phe94 (coloured in cyan) was specially selected as the target of the subsequent alanine-scanning mutagenesis because it exhibited a significant chemical shift change in the pH range of 5–7, although it was classified as group A. The overall structure of NCL is shown in two orientations separated by 180°. The protein surfaces of NAS and NBS are coloured in pale red and pale blue, respectively. (D) Mapping of the residues whose signals were missing in the NMR spectra on the diagram showing the three-dimensional backbone of NCL (coloured in purple). The cysteine residues forming disulphide bonds are shown as ball-and-stick models. Some β-strands are labelled according to our previous paper (Shimizu-Ibuka et al.,25). The backbone of NAS and NBS are coloured in pale red and pale blue, respectively.
Mentions: Next, we measured 1H-15N HSQC NMR spectra at pH 5, 6, and 7 (Fig. 2A and Supplementary Fig. S2A-B). Backbone 1HN and 15N resonances of these spectra were assigned by successively extrapolating the trend of the chemical shift changes to a higher pH. We observed 36 and 33 residues of NAS and NBS, respectively, that exhibited significant differences of more than 0.05 ppm in the 1H chemical shift or 0.5 ppm in the 15N chemical shift between the spectra at pH 3 and 7 (Supplementary Fig. S2C-F). Plotting the normalised chemical shift changes against pH demonstrated that the residues could be classified into two groups (Fig. 2A,B): in group A, most of the chemical shift changes occurred between pH 3 and 5, whereas in group B, most of the chemical shift changes occurred between pH 5 and 7. Because the change from an antagonist to an agonist of NCL occurs between neutral and weakly acidic pH, the residues of group B are more closely related to this function. Because all of these residues are located near His residues in the tertiary structure of NCL (Fig. 2C), the conformational change likely occur upon protonation of the His residues. Moreover, although Phe94 of NBS was classified as group A, it exhibited a significant chemical shift change in the pH range of 5–7. This result suggests that Phe94 is also related to this function.

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