Identification of key neoculin residues responsible for the binding and activation of the sweet taste receptor.
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.
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
Taichi
T
Koizumi
Department of Biotechnology and Agricultural Bioinformatics Research Unit, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
Tohru
T
Terada
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
Ken-ichiro
K
Nakajima
School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan.
Masaki
M
Kojima
1] Laboratory for Biomolecular Structure and Dynamics, RIKEN Quantitative Biology Center (QBiC), Yokohama, Japan [2] Tohoku Medical Megabank Organization, Tohoku University, Sendai, Japan.
Seizo
S
Koshiba
1] Department of Physics, Kansai Medical University, Hirakata, Japan. [2] Present Address: School of Life Sciences Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan.
Yoshitaka
Y
Matsumura
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
Kohei
K
Kaneda
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
Tomiko
T
Asakura
1] Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan [2] Niigata University of Pharmacy and Applied Life Sciences, Akiha-ku, Niigata, Japan.
Akiko
A
Shimizu-Ibuka
1] Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan [2] Kanagawa Academy of Science and Technology, Takatsu-ku, Kawasaki-shi, Kanagawa, Japan.
Keiko
K
Abe
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
Takumi
T
Misaka
D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D016296:Mutagenesis; D019906:Nuclear Magnetic Resonance, Biomolecular; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*
D010940:Plant Proteins; D043562:Receptors, G-Protein-Coupled; C511659:neoculin protein, Curculigo latifolia; C431926:taste receptors, type 1