Studies On The Mechanism Of PEDV 3CL^pro Recognizing The P3 Site Of NEMO By Molecular Dynamic Simulation

Porcine epidemic diarrhea?PED?is a contact intestinal disease in pigs caused by porcine epidemic diarrhea virus?PEDV?.Since the end of 2010,a large-scale outbreak of PED caused by PEDV mutant strains has brought significant economic losses to the global pig industry.At present,there are no drugs that can effectively control the disease in clinic,so the development of anti-PED drugs has become a hotspot.PEDV 3C-like protease(3CL^pro)plays a key role in the proliferation and pathogenesis of PEDV,and it is well conserved in different mutant strains of PEDV.Therefore,PEDV 3CL^pro can be used as an important target for related antiviral drugs.The elucidation of the interaction mechanism between PEDV 3CL^pro and the substrate will provide a theoretical basis for the development of specific drugs targeting PEDV 3CL^pro.Previous laboratory experiments in biology found that,PEDV 3CL^pro recognizes and cleaves NEMO at the position 231?P1?glutamine,a key molecule in the innate immune signaling pathway,when the 229th?P3?glutamine adjacent to the NEMO cleavage site is mutated to arginine?Q-P3-R?,lysine?Q-P3-K?or alanine?Q-P3-A?,mutations of Q-P3-K and Q-P3-R lead to a decrease in the degree of cleavage of substrate NEMO by PEDV3CL^pro compared to WT.Only the Q-P3-A mutation causes NEMO to can't be cut by PEDV 3CL^pro.It was indicated that the mutation of P3 in the substrate would affect the recognition and cleavage of the substrate by PEDV 3CL^pro,suggesting that the P3 site of the substrate plays an important role in the recognition of the substrate by PEDV 3CL^pro.However,the current research on the mechanism of P3 amino acids on the recognition substrate of PEDV 3CL^pro is not clear.In view of this,this paper carried out a study:1.Western blot analysis the characteristics of PDEV 3CL^pro recognizing NEMO P3 positionPrevious literature reports that the P3 locus of the 3CL^pro substrate of coronavirus prefers positively charged amino acids and long side-chain amino acids.In this study,we analyzed the P3 site specificity of PEDV 3CL^pro substrate and found that the amino acid with the highest specificity for P3 is asparagine?N?.Based on this,we constructed two mutants of NEMO 229?QP3?mutations:Q-P3-E and Q-P3-N.the eukaryotic expression plasmids of these two mutants and the wild type and three mutants?Q-P3-A,Q-P3-K,and Q-P3-R?previously constructed in the laboratory were transfected into HEK-293T cells respectively and cutting degree of NEMO by PEDV 3CL^pro were detected by Western blot in different transfection systems.It was found that WT?no charge,long side chain?,Q-P3-K?positive charge,long side chain?,Q-P3-R?positive charge,long side chain?can be cut by PEDV 3CL^pro,and Q-P3-A?no charge,short side chain?cannot be cleaved by PEDV 3CL^pro,consistent with previous laboratory results;in addition,Q-P3-E?negative charge,long side chain?and Q-P3-N?no charge,long side chain?can also be cut by PEDV 3CL^pro,indicating that the PEDV 3CL^pro substrate NEMO P3 position can also be negatively charged amino acids.Further analysis,results can be found that in the cleavage reaction systems,the NEMO P3 position of the substrate has a long side chain characteristic,suggesting that the P3 position of the PEDV 3CL^pro substrate may favor long side chain amino acids.2.Analysis of Free Energy between different mutants of NEMO and PEDV 3CL^proMolecular biology experiments are the cornerstones of molecular dynamics research,and molecular dynamics studies can observe microscopic changes in the motion of the simulation system at the atomic scale.To explore the influence of different mutants of the P3 site of NEMO on the recognition substrate of PEDV 3CL^pro,we used molecular dynamics simulations to analyze Free energy between NEMO wild-type?WT?and five mutants?Q-P3-A,Q-P3-K,Q-P3-R,Q-P3-E,and Q-P3-N?and PEDV 3CL^pro.It was found that the free energy value between Q-P3-A and PEDV 3CL^pro is the largest,indicating that the affinity of the enzyme to the substrate is the lowest in the Q-P3-A system,and the possibility of a cleavage reaction is minimized.The result is consistent with biological experiments confirmed that Q-P3-A is the only one that cannot be cut.3.The existence of ?-sheet structure of NEMO P3 and P2 positions is beneficial to the cutting of PEDV 3CL^proIt has been reported in the literature that P3 preference of the 3CL^pro substrate of the coronavirus can form the?-sheet structure.PEDV also belongs to the Coronaviridae virus.Therefore,we analyzed the secondary structure changes of the substrate P3 and its neighboring amino acids.The results showed that?-sheets formed by P3 and P2 exist in the systems?WT,Q-P3-K,Q-P3-R,Q-P3-E,and Q-P3-N?in which cleavage reaction can occur.However,in the system without cleavage reaction?Q-P3-A?,there is no?-sheet structure formed by P3 and P2,indicating that the presence of the?-sheet structure is conducive to the occurrence of enzymatic reactions.Next,this study analyzed the time for the presence of?-sheet structures on the P3 and P2 substrates in the six systems.It was found that in WT,Q-P3-N and Q-P3-K,the secondary structure of P3 and P2 is always?-sheet,and in Q-P3-R and Q-P3-E,The secondary structure of P3 and P2 are gradually changed from?-sheet to loop or from loop to?-sheet,while in Q-P3-A,the secondary structure of P3 and P2 was quickly changed from?-sheet to loop,indicating that mutation of NEMO P3 position will affect the formation and maintenance of the beta-sheet structure of the P3 and P2 on NEMO.4.Mutations in the NEMO P3 site affect the distance between H41 and C144 of thePEDV 3CL^pro enzyme activity siteAfter confirming that the existence of?-sheet structure of NEMO P3 and P2 sites is beneficial to the cleavage of PEDV 3CL^pro,the specific mechanism of its action was analyzed by molecular dynamics simulation.This study found that when the secondary structure of the P3 and P2 substrates is converted from a?-sheet to a loop,the oxygen conformation of the P2 position of the substrate is reversed from the solvated surface to the direction of the PEDV 3CL^pro enzyme active pocket.Subsequently,a change in the conformation of the oxygen atom at P2 resulted in a change in the conformation of H41 in the amino acid of the S2 pocket;the change in conformation of H41 further led to its relative position to another enzyme site C144 has changed.The first step in the enzymatic reaction of PEDV 3CL^pro is that the nitrogen atom on H41 abstracts the hydrogen atom of-SH on C144.Therefore,the distance between H41 and C144 is critical to the occurrence of enzymatic reactions.Therefore,the heavy atom spacing between PEDV 3CL^pro H41and C144 involved in the enzymatic reaction was further analyzed.It is found that the distance fluctuations between H41 and C144 are significantly different in different systems,indicating that the mutation at position 3 of NEMO affects the distance between H41 and C144 of PEDV 3CL^pro.In addition,we analyzed the correlation between the distance of enzyme activity sites and the cutting degree of NEMO,and found that the correlation coefficient was 0.846,indicating a strong correlation between the two,indicating that the appropriate distance between H41 and C144 in PEDV 3CL^pro is an important factor affecting its cutting of NEMO.