Molecular Dynamic Simulation On Flexible Binding Of Broad-specific Neutralizing Antibody CDR3 With Avian Influenza Virus Receptor Binding Site (RBS) And Structure-Based Rational Design Of Rbs-binding Peptide

Influenza virus evolve and circulate continuesly and widely in the global,causing human respiratory disease which is a serious threat to human health,and it has become a major global public health problem.Every year a large number of people infected with influenza virus,or die from seasonal flu and influenza pandemic.Among them,the threat of avian influenza virus,especially the H5N1 and H7N9 subtype highly pathogenic avian influenza virus has gradually become one of the most serious infectious disease in humans.Due to the high variability of influenza virus,the existing vaccine R&D strategy need to update the epidemic strains every year,and the therapeutic effect of conventional antiviral drugs have also been affected.Therefore,the development of new anti-influenza drugs is an urgent need for prevention and treatment of influenza.The hemagglutinin protein on the influenza virus envelope is the main neutralizing target of antibody recognition,and mediated the envasion to host cells.The receptor binding domain of the hemagglutinin locates at the head region and form a groove structure,and its conserved sites can specifically bind to the sialic acid receptor,which has been used as a potential target in the design of anti-influenza drugs.In recent years,hemagglutinin-based anti-influenza compounds have been developed,but have little practical application.With the development of biochemistry and molecular biology,more and more peptide drugs have been developed and applied to the treatment of major diseases.Compared with the traditional small molecule drugs,peptide drugs with its high activity,high specificity and low toxicity,and other advantages,has become a hot spot for drug development.At present,the efficient and rational design of peptide is the main focus,which has injected new vitality into the development of peptide drugs.Rational structure-based computer aided peptide design predicts the interaction between the peptide and the protein,and designs peptide drugs by using molecular docking,molecular dynamics simulation and theoretical calculation.The first part of the paper focuses on the crystal structure of 13D4 Fab and 13D4 Fab-VN1194 HA complexes determined by our laboratory.The crystal structure of the complex was analyzed and we obtain the interaction and key binding sites of the interface between antigen and antibody.By this method,we found that 13D4 antibody binds to HA protein mainly through its heavy chain CDR3.Comparing the structures of the 13D4 antibody before and after binding the HA protein,we found that the antibody heavy chain CDR3 extend into the groove structure of the receptor binding domain which cause a significant deflection.Next,we reproduced the process of antibody heavy chain CDR3 allosteric fit by molecular dynamics method,and revealed the recognition mechanism of 13D4 antibody to HA protein,and provided the theoretical calculation basis for the peptide drug design.The second part of the paper explores peptide drugs designing based on the binding characteristics of 13D4-HCDR3 and receptor binding domain of HA protein.First,we use Rosetta Pepspec in.peptide design of our study,but the experimental results show that the binding activity of the designed peptides is undetectable.Subsequently,we analyzed the negative results by molecular dynamics simulation and experimental methods,and concluded that the flexibility and conformational freedom of peptide chains is one of the key factors in peptide design.Finally,we established a peptide design method using flexible docking technology and simulated annealing sampling combined with molecular dynamics simulation,and verified the binding activity of the design peptides by experimental methods.In summary,we analyzed the allosteric fitting of HCDR3 using molecular dynamic simulation method and explained the mechanism of recognition of 13D4 antibody at the atomic level.In addition,according to the binding characteristics of the antibody and HA protein,we made a series of attempts in designing of peptide drugs of H5N1 influenza virus,and a set of rational structure-based peptide design method with computer aided technology and bioinformatics has been established.This will provides more theoretical basis for studying the mechanism of neutralizing antibodies of H5 subtypes viruses,and lay the foundation for the future development of anti-influenza broad-spectrum peptide drugs.