| Physical and chemical characteristics of key flexible amino acids are closely related to various physiological properties including protein functional domain composition,molecular recognition and binding mechanism,which play extremely important roles in the normal physiological function of proteins.Qualitative recognition of protein binding interface and quantitative identifications of key flexible residues is one of the key and difficult researching points in studying the physiological function of proteins.Based on the existing massive number of protein structures,one systematic conformation-state differentiation analysis was performed at the molecular level through various biological computing methods.A database for identifying key flexible amino acids on the binding pocket was constructed.Taking the novel coronavirus 2019-nCoV protein as an example,the reliability of key flexible residues identification by computing methods was validated by multiple-mutants construction,in vitro virus infection assays and SPR binding activity experiments.The active molecule Quercetin was validated to possess the ability for completely blocking the binding affinity to human ACE2 protein.The main research contents and innovation points are listed as follows:(1)Based on the novel coronavirus protein structure,we presents an protocol combining the computational biology and multi-scoring strategies to identify key amino acids within the protein binding interface.Firstly,we obtain the direct binding interface between the coronavirus2019-nCoV and human target ACE2 protein by using homology modeling methods.Subsequently,the tolerable mutation prediction was conducted for all 110 amino acids involved in the "protein-protein" binding interface of 2019-nCoV/Spike,and 16 amino acid sites with potential mutational properties were obtained.171 single mutants corresponding to the selected 16 mutated amino acid sites were constructed through biological computational methods.Molecular dynamics simulations were executed to optimize each single-point mutated system to qualitatively analyze the physical-chemical properties of structural region near the mutation site.Comparing analysis of the binding free energies for2019-nCoV-Spike/ACE2 complex between 14 mutant systems and wild-type protein was carried out.Finally,4 key amino acid sites on2019-nCoV protein that can significantly improve the binding ability of coronavirus to human ACE2 protein was identified.(2)One protocol to identify the key flexible amino acid sites on the Spike domain of coronavirus were obtained based on systematical researches combining synergistic enhancement effects for multi-mutant system and vitro cell infective validation experiments.Firstly,31 potential amino acid sites were identified by analyzing the2019-nCoV-Spike/ACE2 protein-protein binding interface and integrating data information from the CNCB-NGDC coronavirus databank,respectively.Subsequently,binding affinities for 589 single-point mutation systems of 2019-nCoV-Spike/ACE2 were calculated by using molecular dynamics simulation methods,and 8 mutations with higher binding energies were picked out.Random computational modeling based on the 8 mutations were carried out to obtain 184 multiple-mutants of 2019-nCoV-Spike protein.Binding energies for each system was calculated.Finally,virus infection assays of 6 multiple-mutants comparing with wild-type coronavirus further confirmed collaborative affects and revealed the significant correlation between binding free energies and viral infectivity.These findings support continuing surveillance of 2019-nCoV coronavirus mutations to aid developments of drug and vaccines.Moreover,binding energies analysis of all 773 mutants indicated that Y449 R and S494 R played as the key-point residues which possess collaborative enhancement effects to other mutants.(3)One protocol for identifying anti-coronavirus ligands was proposed combing the network pharmacology and semi-flexible docking based virtual screening technologies.SPR experiments were conducted to verify the blocking ability of the molecule quercetin to coronavirus2019-nCoV with that of ACE2 protein.In this study,based on the network pharmacology method,41 kinds of virus-related active components were identified from 499 traditional Chinese medicines and29384 components in the TCMSP Chinese medicine database.The identified key flexible residues were regarded as flexible parameters for carrying out virtual screening from the Chinese medicine database.Ingredients including puerarin,kaempferol and quercetin possessing existed good clinical activities were identified.In vitro SPR activity experiments were executed and verified the binding ability of coronavirus to the selected 3 compounds.It was found that the quercetin can completely block the binding ability between the 2019-nCoV-Spike and the ACE2 receptor.At present,quercetin has been tested with good effects in inhibiting virus infecting animal experiments including mice and rabbits,and the following Phase I clinical trials will be lunched subsequently.(4)One comprehensive evaluation method for systematically identifying key flexible amino acids in binding pockets of protein molecules was proposed,and the KeyFlex database was developed.Based on the existing 156,800 three-dimensional crystal protein structure s,41754 clusters of identical proteins with different structures were obtained by 95% sequence similarity clustering.The active molecules were determined,and the 6310 subtype protein structural clusters containing >3 protein chains with different structures were picked out.Characterization factors variation of physicochemical and structural parameters including RMSD,B-factor,mutation site,residue solvent accessibility,steric clash and dihedral angles were calculated to investigate the nature of protein flexibility.Systematical top scoring ranked residues in protein binding pocket were identified as the key flexible residues.The database KeyFlex containing key flexible residue for all existing crystal protein will be constructed. |
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