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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

Crystal structure of NCL at pH 7.4 (PDB ID: 2D04).Neoculin is a heterodimeric protein composed of the neoculin acidic subunit (NAS) and neoculin basic subunit (NBS). NAS is indicated with a pale red ribbon, whereas NBS is indicated with a pale blue ribbon. The cysteine residues forming disulphide bonds are represented as ball-and-stick models and coloured in yellow. The histidine residues are shown with a stick model. NBS His11, a primary pH sensor of NCL, is coloured in green, whereas NAS His36 and NBS His14, which are partially responsible for the pH-dependency of NCL, are coloured in pale green. All structural images (with the exception of Fig. 5) were generated using Discovery Studio Visualizer 3.0 (Accelrys Software Inc).
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f1: Crystal structure of NCL at pH 7.4 (PDB ID: 2D04).Neoculin is a heterodimeric protein composed of the neoculin acidic subunit (NAS) and neoculin basic subunit (NBS). NAS is indicated with a pale red ribbon, whereas NBS is indicated with a pale blue ribbon. The cysteine residues forming disulphide bonds are represented as ball-and-stick models and coloured in yellow. The histidine residues are shown with a stick model. NBS His11, a primary pH sensor of NCL, is coloured in green, whereas NAS His36 and NBS His14, which are partially responsible for the pH-dependency of NCL, are coloured in pale green. All structural images (with the exception of Fig. 5) were generated using Discovery Studio Visualizer 3.0 (Accelrys Software Inc).

Mentions: Neoculin (NCL) is a sweet protein isolated from the edible fruit of the tropical plant Curculigo latifolia. NCL exhibits a taste-modifying activity that converts sourness to sweetness. NCL elicits a slightly sweet taste on its own and elicits a strongly sweet taste in the presence of an acidic solution20. This protein is a heterodimer composed of an acidic subunit (neoculin acidic subunit, NAS) of 113 amino acid residues and a basic subunit (neoculin basic subunit, NBS) of 114 amino acid residues. Although the two subunits share 77% amino acid identity, they differ in terms of their isoelectric points; the pI for NAS is between pH 4.0 and 6.0, whereas that for NBS is between pH 7.5 and 9.511. They form a clamshell-like structure and are connected by two disulphide bonds (Fig. 1). We previously revealed that NCL acted as an hT1R2-hT1R3 antagonist at neutral pH and as an agonist at acidic pH; histidine residues were responsible for this pH-dependent functional change21. Of two and three NAS and NBS histidine residues, we demonstrated that NBS His11 functioned as a primary pH sensor22. Therefore, we inferred that protonation of the histidine residues caused a conformational change in NCL and influenced its binding to the receptor21.


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)

Crystal structure of NCL at pH 7.4 (PDB ID: 2D04).Neoculin is a heterodimeric protein composed of the neoculin acidic subunit (NAS) and neoculin basic subunit (NBS). NAS is indicated with a pale red ribbon, whereas NBS is indicated with a pale blue ribbon. The cysteine residues forming disulphide bonds are represented as ball-and-stick models and coloured in yellow. The histidine residues are shown with a stick model. NBS His11, a primary pH sensor of NCL, is coloured in green, whereas NAS His36 and NBS His14, which are partially responsible for the pH-dependency of NCL, are coloured in pale green. All structural images (with the exception of Fig. 5) were generated using Discovery Studio Visualizer 3.0 (Accelrys Software Inc).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Crystal structure of NCL at pH 7.4 (PDB ID: 2D04).Neoculin is a heterodimeric protein composed of the neoculin acidic subunit (NAS) and neoculin basic subunit (NBS). NAS is indicated with a pale red ribbon, whereas NBS is indicated with a pale blue ribbon. The cysteine residues forming disulphide bonds are represented as ball-and-stick models and coloured in yellow. The histidine residues are shown with a stick model. NBS His11, a primary pH sensor of NCL, is coloured in green, whereas NAS His36 and NBS His14, which are partially responsible for the pH-dependency of NCL, are coloured in pale green. All structural images (with the exception of Fig. 5) were generated using Discovery Studio Visualizer 3.0 (Accelrys Software Inc).
Mentions: Neoculin (NCL) is a sweet protein isolated from the edible fruit of the tropical plant Curculigo latifolia. NCL exhibits a taste-modifying activity that converts sourness to sweetness. NCL elicits a slightly sweet taste on its own and elicits a strongly sweet taste in the presence of an acidic solution20. This protein is a heterodimer composed of an acidic subunit (neoculin acidic subunit, NAS) of 113 amino acid residues and a basic subunit (neoculin basic subunit, NBS) of 114 amino acid residues. Although the two subunits share 77% amino acid identity, they differ in terms of their isoelectric points; the pI for NAS is between pH 4.0 and 6.0, whereas that for NBS is between pH 7.5 and 9.511. They form a clamshell-like structure and are connected by two disulphide bonds (Fig. 1). We previously revealed that NCL acted as an hT1R2-hT1R3 antagonist at neutral pH and as an agonist at acidic pH; histidine residues were responsible for this pH-dependent functional change21. Of two and three NAS and NBS histidine residues, we demonstrated that NBS His11 functioned as a primary pH sensor22. Therefore, we inferred that protonation of the histidine residues caused a conformational change in NCL and influenced its binding to the receptor21.

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