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Abstract
Sucrose provide sweetness and energy when it binds to both the Venus fly trap domains (VFD) of heterodimeric sweet taste receptor (T1R2/T1R3),1 while non-caloric sweeteners (Sucralose, Aspartame, Neotame, Saccharin, Acesulfame-K (Ace-K), Suosan, SC-45647, Fructose, and D-Tryptophan) bind only at the VFD of T1R2 (VFD2) to provide high-intensity sweetness.2 Here, we address the binding mechanism of various steviol glycosides (Rebaudioside B (RebB), RebM, RebD, Rubusoside (Rubu), RebC), artificial sweeteners (Neohesperidin dihydrochalcone (NHDC), acesulfame K, S-819, Amiloride, and Perillartine), and negative allosteric modulator (Lactisole) at four distinct binding sites, VFD2, VFD3, transmembrane domain 2 (TMD2) and TMD3 by performing binding experiments and computational docking studies. Our docking results find multiple binding sites for the tested ligands including the radio labelled ligands, which provides an explanation of the mixed data of the radio-ligand binding experiments. We predict different preferred binding sites depending on ligand modifications of steviol glycosides. Thus, Rubu binds best at VFD3, while RebB binds best at TMD3, while the others prefer VFD2. We also observed G protein-coupled receptor (GPCR) allostery using the label free Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) method. We show experimentally that the C20 carboxy terminus of the Gα protein can bind to the intracellular region of either TMD2 or TMD3, which can alter GPCR affinity to the high-affinity state for steviol glycosides. These studies provide a mechanistic understanding of the structure and function of this heterodimeric sweet taste receptor that can guide rational structure-based design of novel non-caloric sweeteners aimed at enabling lower sugar usage levels while retaining the sweet taste. This may provide the basis for novel therapeutic applications for treatment of obesity and related metabolic dysfunctions such as diabetes.
Supplementary materials
Title
Supporting Information
Description
Competition binding experiments of various ligands with [3H]-Rebaudioside B, Competition binding experiments using [3H]-Lactisole as radioligand against Ligand, Competition binding experiments using [3H]-Perillartine as radioligand, The free energy of the binding of steviol glycosides to heterodimeric sweet taste receptor (T1R2/T1R3) using molecular mechanics with generalized Born and surface area solvation (MM/GBSA), Overview of FLOWER, FLOWER binding response, Normal FLOWER binding response curves, Effect of GTP on the binding of [3H]-Rebaudioside B, Kinetic binding using [3H]-Lactisole as radioligand, Effect of GDP on the binding of [3H]-Lactisole Concentration, Effect of GTP on the binding of [3H]-Lactisole, Kinetic binding using [3H]-Perillartine as radioligand, Effect of GDP on the binding of [3H]-Perillartine, Effect of GTP on the binding of [3H]-Perillartine, Kinetic binding using [14C]-sucrose as radioligand, Competition binding curve of variable concentration of NHDC against fixed concentration of [14C]-sucrose, All putative binding sites of steviol glycosides obtained through SiteMap study.
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