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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.


Comparison of the representative structures of the MD simulations under weakly acidic and neutral pH conditions with the crystal structure at pH 7.4 (PDB ID: 2D04).Structures are aligned with respect to NBS. NAS and NBS of the MD structure at pH 5, the MD structure at pH 7, and the crystal structure are coloured light red, dark red, light green, dark green, light blue, and dark blue, respectively. The structural images were generated using UCSF Chimera39.
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f5: Comparison of the representative structures of the MD simulations under weakly acidic and neutral pH conditions with the crystal structure at pH 7.4 (PDB ID: 2D04).Structures are aligned with respect to NBS. NAS and NBS of the MD structure at pH 5, the MD structure at pH 7, and the crystal structure are coloured light red, dark red, light green, dark green, light blue, and dark blue, respectively. The structural images were generated using UCSF Chimera39.

Mentions: We previously hypothesised that NCL changes its conformation from closed to open as the pH decreases25. This hypothesis was based on the results of molecular dynamics (MD) simulations performed on NCL under neutral and acidic pH conditions. In the open conformation, the two subunits (NAS and NBS) comprising NCL were expected to partially dissociate from each other. However, the residues located in the interface between the two subunits did not show significant chemical shift changes in our NMR analysis between pH 5 and 7 (Fig. 2C). Moreover, only a limited number of residues showed significant chemical shift changes at weakly acidic pH in the entire NCL structure. The electrostatic repulsive interactions between the subunits may have been overestimated in the previous MD simulations because they used an implicit solvent model to reduce the computational cost. Therefore, we performed MD simulations for NCL under neutral and weakly acidic conditions with an explicit water model for an extended period of time (2 μs). Large conformational changes were not observed during the simulations, except for the loop between β6 and β7 of NBS (Fig. 5). The conformation of this loop significantly deviated from the conformation in the crystal structure in both simulations, which is consistent with the finding that the NMR signals of the residues in this loop were missing due to conformational exchange regardless of pH. Furthermore, SAXS analysis using NCL under neutral and acidic pH conditions indicated no difference in molecular size (Fig. 6). Therefore, NCL undergoes only local conformational changes at weakly acidic pH. Because this local change activates the receptor, the residues responsible for the local conformational change and pH-dependency should be located close to the receptor in the complex structure.


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)

Comparison of the representative structures of the MD simulations under weakly acidic and neutral pH conditions with the crystal structure at pH 7.4 (PDB ID: 2D04).Structures are aligned with respect to NBS. NAS and NBS of the MD structure at pH 5, the MD structure at pH 7, and the crystal structure are coloured light red, dark red, light green, dark green, light blue, and dark blue, respectively. The structural images were generated using UCSF Chimera39.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Comparison of the representative structures of the MD simulations under weakly acidic and neutral pH conditions with the crystal structure at pH 7.4 (PDB ID: 2D04).Structures are aligned with respect to NBS. NAS and NBS of the MD structure at pH 5, the MD structure at pH 7, and the crystal structure are coloured light red, dark red, light green, dark green, light blue, and dark blue, respectively. The structural images were generated using UCSF Chimera39.
Mentions: We previously hypothesised that NCL changes its conformation from closed to open as the pH decreases25. This hypothesis was based on the results of molecular dynamics (MD) simulations performed on NCL under neutral and acidic pH conditions. In the open conformation, the two subunits (NAS and NBS) comprising NCL were expected to partially dissociate from each other. However, the residues located in the interface between the two subunits did not show significant chemical shift changes in our NMR analysis between pH 5 and 7 (Fig. 2C). Moreover, only a limited number of residues showed significant chemical shift changes at weakly acidic pH in the entire NCL structure. The electrostatic repulsive interactions between the subunits may have been overestimated in the previous MD simulations because they used an implicit solvent model to reduce the computational cost. Therefore, we performed MD simulations for NCL under neutral and weakly acidic conditions with an explicit water model for an extended period of time (2 μs). Large conformational changes were not observed during the simulations, except for the loop between β6 and β7 of NBS (Fig. 5). The conformation of this loop significantly deviated from the conformation in the crystal structure in both simulations, which is consistent with the finding that the NMR signals of the residues in this loop were missing due to conformational exchange regardless of pH. Furthermore, SAXS analysis using NCL under neutral and acidic pH conditions indicated no difference in molecular size (Fig. 6). Therefore, NCL undergoes only local conformational changes at weakly acidic pH. Because this local change activates the receptor, the residues responsible for the local conformational change and pH-dependency should be located close to the receptor in the complex structure.

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.