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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.


Probable model for the taste-modifying activity of NCL.Cartoon representation of the possible model. At neutral pH, NCL binds to the human sweet receptor and slightly activates it (Left). At acidic pH, the binding mode is locally altered by protonation of the histidine residues of NCL, leading to the strong activation of the receptor (Right).
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f7: Probable model for the taste-modifying activity of NCL.Cartoon representation of the possible model. At neutral pH, NCL binds to the human sweet receptor and slightly activates it (Left). At acidic pH, the binding mode is locally altered by protonation of the histidine residues of NCL, leading to the strong activation of the receptor (Right).

Mentions: The residues important for the pH-independent affinity were located separately from the residues responsible for the pH-dependency in the tertiary structure. Therefore, NCL interacts with the receptor through two interfaces: (1) the pH-independent affinity interface, whose interaction with the receptor is not influenced by the pH change and determines the affinity for the receptor, and (2) the pH-dependent activation interface, whose interaction with the receptor is altered by the pH change and induces the activation/inactivation of the receptor. In the pH-dependent activation interface, hydrogen bonds between the histidine residues of NCL and the receptor atoms suppress the activation of the receptor at neutral pH. Protonation of the histidine residues disrupts the hydrogen bonds and locally alters the structure of the interface, leading to the activation of the receptor at acidic pH (Fig. 7).


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)

Probable model for the taste-modifying activity of NCL.Cartoon representation of the possible model. At neutral pH, NCL binds to the human sweet receptor and slightly activates it (Left). At acidic pH, the binding mode is locally altered by protonation of the histidine residues of NCL, leading to the strong activation of the receptor (Right).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: Probable model for the taste-modifying activity of NCL.Cartoon representation of the possible model. At neutral pH, NCL binds to the human sweet receptor and slightly activates it (Left). At acidic pH, the binding mode is locally altered by protonation of the histidine residues of NCL, leading to the strong activation of the receptor (Right).
Mentions: The residues important for the pH-independent affinity were located separately from the residues responsible for the pH-dependency in the tertiary structure. Therefore, NCL interacts with the receptor through two interfaces: (1) the pH-independent affinity interface, whose interaction with the receptor is not influenced by the pH change and determines the affinity for the receptor, and (2) the pH-dependent activation interface, whose interaction with the receptor is altered by the pH change and induces the activation/inactivation of the receptor. In the pH-dependent activation interface, hydrogen bonds between the histidine residues of NCL and the receptor atoms suppress the activation of the receptor at neutral pH. Protonation of the histidine residues disrupts the hydrogen bonds and locally alters the structure of the interface, leading to the activation of the receptor at acidic pH (Fig. 7).

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.