Research On The Molecular Mechanism Of Umami Recognition By T1R1-T1R3

Taste receptor T1R1-T1R3 as an umami receptor can response to several ligands（natural substances）,e.g.,L-glutamate and 5’-ribonucleotides etc.However,the crystal structure of hT1R1-T1R3 has not been reported,and the detailed molecular mechanism is still unknown.In this study,homology modeling,molecular docking and molecular dynamics（MD）simulations were used to construct five umami ligands-bound T1R1-T1R3 complexes based on fish taste receptor T1R2a-T1R3（PDB id:5X2M）and ligands including L-glutamic acid salt（MSG）,sodium succinate（WSA）,disodium 5’-guanylate（GMP）,disodium 5’-inosinate（IMP）and beefy meaty peptide（BMP）.Moreover,the effect of ligands binding on T1R1-T1R3 dynamics and molecular mechanism of umami recognition were further explored and analyzed at an atomistic-level.The main results are as follows:（1）The human T1R1-T1R3 heterodimer structure model was constructed by homologous modeling with the target of the amino acid sequence of the extracellular Venus Flytrap domain of human T1R1 and T1R3.The homology model was fish T1R2a-T1R3,and the human T1R1 and T1R3 subunits share sequence identity of33.03%and 33.87%with the corresponding subunit of fish sweet receptor,respectively.On the other hand,based on the amino acid sequence and structure of the receptors including taste receptors and metabotropic glutamate receptors,the evolutionary analysis results showed that the T1R1-T1R3 had higher homology with fish T1R2a-T1R3 compared to mGluR1.Structure overlay analysis of T1R1-T1R3,T1R2a-T1R3 and mGluR1 were performed based on their C_αatoms.The results indicated the RMSD was equal 3.2?for T1R1-T1R3 and mGluR1,but the RMSD is equal 2.4?for T1R1-T1R3 and T1R2a-T1R3.These indicated T1R1-T1R3 structural is reasonable based on T1R2a-T1R3 as a template.（2）Five umami ligand-bound T1R1-T1R3 complexes were constructed,and their ligand included L-glutamic acid salt（MSG）,sodium succinate（WSA）,disodium5’-guanylate（GMP）,disodium 5’-inosinate（IMP）and beefy meaty peptide（BMP）.Structure overlay analysis of T1R2a-T1R3 and mGluR1 was performed based on their C_αatoms,and C_α-COO^-and C_α-NH₃⁺group of ligands in all complexes reside were found in a same pocket,and they lied in a similar orientation.Therefore,the C_αatomic coordinates of the ligand（L-Glutamine）in the template T1R2a-T1R3 were used as the docking center,and the molecular docking was performed in a cubic box of 30?size.Finally,the ligand-T1R1-T1R3 complex after molecular docking were used for the initial structure of molecular dynamics simulation.（3）The effects of ligands binding on T1R1-T1R3 dynamics and molecular mechanism of umami recognition explored and analyzed.For each ligand-T1R1-T1R3and without ligand-T1R1-T1R3（apo）,their dynamics analysis was performed by 100ns molecular simulation.The RMSD results indicated that ligand binding primarily led to the movement of a subunit（LB2）on T1R1.Some key residues in the T1R1which play essential roles in recognizing the ligands were identified.These identified residues were critical for MSG,IMP and GMP recognitions,and they were supported by former site-directed mutagenesis studies.Further ligand network analysis showed that the two binding sites both existed in T1R1,namely binding site A and binding site B.MSG and WSA can bind in the binding site A,and BMP bind to the binding site B.However,GMP and IMP occupied two binding sites.Finally,for each binding site,the molecular dynamics trajectories of all systems were projected by two reaction coordinates,namely,RMSD and radius of gyration（Rg）.The results indicated that the Apo system can be used as a ground state at the binding site A,and five umami ligands can be divided into two types.MSG,GMP and IMP,promoted the closure of binding site A,but WSA and BMP,promoted the opening of binding site A.On the other hand,the electrostatic potential calculation（Adaptive Poisson-Boltzmann Solver,APBS）was performed on T1R1,and the results showed that the binding site B contained more positively charged environments than the binding site A.Taken together,a more accurate homology model of T1R1-T1R3 was constructed by using a new template structure different from the former theoretical studies（mGluR）.In addition,some key residues,which play essential roles in recognizing the ligands,were identified.Two binding sites in T1R1 were found.More interestingly,,the ligand could bind one or both of these two binding sites depending on their molecular size and chemical properties.Moreover,they can also regulate the closing and opening motions of T1R1.This work revealed molecular mechanism of umami recognition at an atomistic-level,and the result further guides to design more umami ligands.