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

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.

De novo transcriptome analysis and comparative expression profiling of genes associated with the taste-modifying protein neoculin in Curculigo latifolia and Curculigo capitulata fruits

“…Four residues are responsible for the binding and activation of the human sweet receptor: Arg-48, Tyr-65, Val-72, and Phe-94 [26]. Although Tyr-65 and Val-72 were identi ed in the C_9931 series, Leu-48 and Val-94 were missing.…”

“…The two subunits share 77% identity at the protein level [18]. Several essential amino acids that are responsible for the taste-modifying properties of neoculin have been identi ed: His-11 in NBS is responsible for the pH-dependent taste-modifying activity of neoculin [25], and Arg-48, Tyr-65, Val-72, and Phe-94 function in the binding and activation of human sweet taste receptors [26]. Changes in the tertiary structure of the subunits at these residues are thought to contribute to the taste-modifying properties of neoculin [27,28].…”

De novo transcriptome analysis and comparative expression profiling of genes associated with the taste-modifying protein neoculin in Curculigo latifolia and Curculigo capitulata fruits

“…Four residues are responsible for the binding and activation of the human sweet receptor: Arg48, Tyr65, Val72, and Phe94 [26]. Although Based on this alignment, we investigated the amino-acid residue substitution in each region compared with each reference sequence: NBS or NAS (Additional File 8).…”

De novo transcriptome analysis and comparative expression profiling of genes associated with the taste-modifying protein neoculin in Curculigo latifolia and Curculigo capitulata fruits

“…The two subunits share 77% identity at the protein level [ 18 ]. Several essential amino acids that are responsible for the taste-modifying properties of neoculin have been identified: His-11 in NBS is responsible for the pH-dependent taste-modifying activity of neoculin [ 25 ], and Arg-48, Tyr-65, Val-72, and Phe-94 function in the binding and activation of human sweet taste receptors [ 26 ]. Changes in the tertiary structure of the subunits at these residues are thought to contribute to the taste-modifying properties of neoculin [ 27 , 28 ].…”