Positive Charges on the Surface of Thaumatin Are Crucial for the Multi-Point Interaction with the Sweet Receptor

Abstract

Thaumatin, an intensely sweet-tasting protein, elicits sweet taste with a threshold of only 50 nM. Previous studies from our laboratory suggested that the complex model between the T1R2-T1R3 sweet receptor and thaumatin depends critically on the complementarity of electrostatic potentials. In order to further validate this model, we focused on three lysine residues (Lys78, Lys106, and Lys137), which were expected to be part of the interaction sites. Three thaumatin mutants (K78A, K106A, and K137A) were prepared and their threshold values of sweetness were examined. The results showed that the sweetness of K106A was reduced by about three times and those of K78A and K137A were reduced by about five times when compared to wild-type thaumatin. The three-dimensional structures of these mutants were also determined by X-ray crystallographic analyses at atomic resolutions. The overall structures of mutant proteins were similar to that of wild-type but the electrostatic potentials around the mutated sites became more negative. Since the three lysine residues are located in 20-40 Å apart each other on the surface of thaumatin molecule, these results suggest the positive charges on the surface of thaumatin play a crucial role in the interaction with the sweet receptor, and are consistent with a large surface is required for interaction with the sweet receptor, as proposed by the multipoint interaction model named wedge model.

Keywords: SHELXL; atomic resolution; electrostatic potential; lysine; positive charge; sweet-tasting protein.

Figure 1

Comparison of normalized B-factor values among thaumatin mutants. Histograms of Normalized B-factor (B-factor of each residue/B-factor whole average) with the residue number. (A) Main-chain for K78A (red), K106A (blue), K137A (green), and recombinant thaumatin (black). (B) side-chain for K78A (red), K106A (blue), K137A (green), and recombinant thaumatin (black) thaumatin. The structural data of recombinant thaumatin was from PDB entry 3al7 (Masuda et al., 2011a).

Figure 2

Electrostatic potential surface of thaumatin mutants. The electrostatic potentials of recombinant thaumatin (A), K78A (B), K106A (C), and K137A (D) are shown in a surface model from acidic (red) to basic (blue). The residues at positions 67, 78, 82, 106, and 137 are indicated in arrows. The mutated residues are indicated in each panel. Molecular models were generated with PyMOL. The structural data of recombinant thaumatin was from PDB entry 3al7 (Masuda et al., 2011a).

Figure 3

The wedge complex of thaumatin mutants with the T1R2-T1R3 receptor. (A) A view of the complex between D21N thaumatin (Masuda et al., 2016). The model of the receptor is shown as a line representation (blue) of the backbone whereas the model of thaumatin is shown as a neon representation of the backbone (gold). The side chains of the key basic residues of thaumatin chosen to optimize the complex are represented as thick blue neons. The corresponding side chains of the acidic residues of the receptor are represented as magenta neons. (B) Enlargement of the interaction zone surrounded by green dots in panel (A). This view shows the side of the sweet protein in contact with the receptor. Receptor residues are labeled with the prefix r2_ when belonging to the T1R2 protomer and with r3_ when belonging to the T1R3 protomer respectively. (C) Corresponding enlargement of the putative complexes of K78A, K106A and K137A-thaumatin with the T1R2-T1R3 receptor. The alanines are shown with green labels. It is easy to see that three important positive-negative interactions disappear. Models were built with MOLMOL (Koradi et al., 1996).