Research On The Mechanism Of Interaction Of Two Sweeteners With The Sweet Taste Receptor And Their Molecular Evolution Based On Receptor Activation

Sweet taste,one of the five basic taste sensation of primates,provides dietary perference and selection for primates,which is crucial for their fitness and survival.Sweet taste perception is mediated by the broadly active class C G protein-coupled receptor(GPCR)-the sweet taste receptor heterodimer of Tas1r2/Tas1r3(T1R2/T1R3)on the cell membrane of taste bud cells located in the mouth.When sweetener interacts with the sweet taste receptor,its conformation changes and thus the sweet taste receptor is activated.Until now,enormous progress has been made towards human sweet taste receptor.However,information about primates other than human is still scarce,and the structure-function relationship of T1R2s/T1R3 s in primates,especially the molecular basis for their species-dependent sweet taste,has not been well understood until now.Firstly,based on the previous reports,it is konwn that the sweet perception in primates shows species-dependent properties.Therefore,a comprehensive sequence,structural and functional analysis of sweet taste receptors in primates was performed to elucidate the molecular determinants mediating the functional diversity and evolution as well as their species-dependent sweet taste recognition.Our results reveal distinct taxonomic distribution and significant characteristics(interaction,coevolution and epistasis)of specific key functionrelated residues,which are reinforced by the sequence similarity networks and variation/conservation in phylogenetic analysis.These characteristics could partly account for the previously reported behavioral results of taste perception in primates.Secondly,in order to elucidate the interaction mechanism between human sweet taste receptor and the natural sweetener stevioside(STV),various residues(T1R2: S40,K65,I67,Y103,D142,S144,S165,P277,D278,E302,S303,D307,R378,R383;T1R3: R52,E105)on the human sweet taste receptor T1R2/T1R3 were site-directed mutated based on the previously reported binding patterns of small molecular sweet compounds such as aspartame and tryptophan in human sweet taste receptor,and the activities of these mutants at the cellular level were assayed to show their function.According to the above experimental results,molecular docking simulation of stevioside in human sweet taste receptor was conducted,and the sites of mutants with decreased activity toward stevioside were used as anchor points in docking(T1R2:S40,K65,Y103,D142,S165).Based on functional experiments and molecular simulations,we identified some key residues related to the activities of stevioside within the binding pocket in sweet taste receptor(residues with reduced activities: S40,N44,Y103,D142,S144,S165,P277 and D278 in T1R2;residues with complete loss activities: K65,I67 and R383 in T1R2 and E105 in T1R3).These residues play important roles for the ligand recognition of sweet taste receptor and the intensity of activity of stevioside via interactive forces such as hydrogen bonds and hydrophobic interaction.Next,based on the sequence analysis in chapter two,it is found to be interesting that the prosimians Lemuriformes species,which were reported to have no sensitivity to aspartame,could be proposed to be aspartame tasters.By functional characterization of the sweet taste receptor of a species of prosimians Lemuriformes,the coquerel’s sifaka and two single-site mutants on its T1R2,this hypothesis was verified.These results promote a better understanding for the diversity,function and evolution of sweet taste receptors in primates.With a comprehensive sequence and structure analysis of sweet taste receptors in primates and functional investigations at a cell level,this study elucidated the molecular mechanism of the diversity,function and evolution of sweet taste receptors in primates,and then explored the mechanism of their interaction with the sweet taste receptor.This study not only lays a foundation for the design of new superior sweeteners in the future,but also provides new insights for the function and evolution research of sweet taste receptors in primates.