Sweet Protein Allosteric Binding and Activation of the Human T1R2/R3 Sweet Receptor: A Simulation Model Validated by in Vitro Receptor Activation Assay

Sweet proteins trigger sweet taste perception through interactions with the human T1R2/R3 sweet taste receptor. To date, relatively few proteins have been identified as causing sweet taste perception, and the four most studied proteins: monellin, brazzein, thaumatin, and honey truffle active component (HT-AC), have minimal sequence homology or structural similarities aside from positively charged surface sites. Sweet taste perception has also been found to be readily perturbed by minor changes in the protein structure, such as natural isoforms inherent to heterologous expression of the protein, and synthetic amino acid substitutions. This study uses ab initio rigid-body docking to predict the interactions of known sweet proteins and variants with a recently resolved cryo-EM structure of the T1R2/R3 sweet taste receptor, incorporating comparative analyses between apo-, holo-, and a potentially transient conformation of the receptor. HT-AC mediated activation of the sweet taste receptor is confirmed by in vitro cell-based assays, and results from in silico docking of various sweet proteins are used to derive additional insights regarding sweet taste perception. Perturbations of HT-AC due to naturally occurring post-translational modifications and synthetic modifications are evaluated using in vitro and in silico methods to determine robustness of the interaction between T1R2/R3 and sweet proteins with primary focuses on HT-AC.