Key Amino Acid Residues Involved in Multi-Point Binding Interactions between Brazzein, a Sweet Protein, and the T1R2–T1R3 Human Sweet Receptor

Abstract

The sweet protein brazzein activates the human sweet receptor, a heterodimeric G-protein coupled receptor (GPCR) composed of subunits T1R2 and T1R3. In order to elucidate the key amino acid(s) responsible for this interaction, we mutated residues in brazzein and each of the two subunits of the receptor. The effects of brazzein mutations were assayed by a human taste panel and by an in vitro assay involving receptor subunits expressed recombinantly in human embryonic kidney cells; the effects of the receptor mutations were assayed by the in vitro assay. We mutated surface residues of brazzein at three putative interaction sites: Site 1 (Loop43), Site 2 (N-and C-terminus and adjacent Glu36, Loop33), and Site 3 (Loop9-19). Basic residues in Site 1 and acidic residues in Site 2 were essential for positive responses from each assay. Mutation of Y39A (Site 1) greatly reduced positive responses. A bulky side chain at position 54 (Site 2), rather than a side chain with hydrogen bonding potential, was required for positive responses as was the presence of the native disulfide bond in Loop 9-19 (Site 3). Results from mutagenesis and chimeras of the receptor indicated that brazzein interacts with both T1R2 and T1R3 and that the Venus fly trap module of T1R2 is important for brazzein agonism. With one exception, all mutations of receptor residues at putative interaction sites predicted by wedge models failed to yield the expected decrease in the brazzein response. The exception, hT1R2:R217A-hT1R3, which contained a substitution in lobe 2 at the interface between the two subunits, exhibited a small selective decrease in brazzein activity. However, because the mutation was found to increase the positive cooperativity of binding by multiple ligands proposed to bind both T1R subunits (brazzein, monellin, and sucralose) but not those that bind to a single subunit (neotame and cyclamate), we suggest that this site in involved in subunit-subunit interaction rather than direct brazzein binding. Results from this study support a multipoint interaction between brazzein and the sweet receptor by some mechanism other than the proposed wedge models.

Allosteric Modulation of Family C G-Protein-Coupled Receptors: from Molecular Insights to Therapeutic Perspectives

“…Subsequently, Koizumi et al (2007), using human/mouse chimeric constructs expressed in HEK cells and intracellular calcium mobilization assays, have demonstrated that the protein neoculin, which tastes sweet to man but not mice, is indeed received by the Nterminal domain of T1R3. However, the wedge model has now been found not to be consistent with the results of mutational analysis, at least in the case of brazzein (Assadi- Porter et al, 2010). On the other hand, Jiang et al (2004), also by using human/mouse chimeras and by performing site-directed mutagenesis experiments, came to the conclusion that the cysteine-rich region of human T1R3 determines the responses to intensely sweet proteins such as brazzein or monellin and might be part of their binding site (Fig.…”

Glucose sensing and signalling; regulation of intestinal glucose transport

“…There are also structural and sequential variations of the sweet taste receptor in various species (44) . Recent investigations demonstrate different functional roles of the subunits as well as the presence of discrete sites responsible for binding ligands of different chemical structures (45) . In lingual epithelium, the key elements of taste transduction pathways are a-, b-and g-subunits of gustducin, phospholipase Cb2 and transient receptor potential melastatin 5, a Ca 2 + -activated cation channel (46)(47)(48) .…”

The Effect of Mutation in Brazzein Deduced from Mutational Sites and Sequence Carbon Content

“…The present work studied the hydrophobicity through carbon content on a number of brazzein mutants previously reported with focus on the two critical regions of the molecule suggested to be responsible for its sweetness [1,8,9,15]. As revealed in the results, the two regions with residues 29-33 and 39-43 including residue 36 between these segments, exists a remarkable variation between brazzein mutants with the increased sweetness taste mainly at residues 29, 31 and 41 and mutants with reduced sweetness taste at residues 30, 33, 36 and 43.…”

“…Brazzein mutants used in this study are based on mutagenesis studies in brazzein's two important regions previously reported as determinants of sweetness taste of the protein at residues 29-33 and 39-43, plus residue 36 [1,8,9]. The residues specifically examined were in position number 29, 30, 31, 33, 36, 41 and 43.…”

“…Mutation of amino acid sequence can occur with or without effects on protein stability and function. Previous studies on mutations in brazzein have reported some effects on its sweet-tasting properties whereby some mutations have resulted in largest increase in sweetness, while other mutations have basically caused brazzein to be tasteless [1,[8][9][10][11]. The studies have examined the role of the surface charge of the molecule as being an important for brazzein's sweetness taste.…”