Quantitative Analysis Of The Substrate Specificity Of Human Rhinovirus 3C Protease And Exploration Of Its Substrate Recognition Mechanisms

Human rhinovirus(HRV)3C protease is a cysteine protease encoded by human rhinovirus.Inhibitors against HRV 3C protease are one of the important means to prevent infection by small RNA viruses.At the same time,due to its high proteolytic activity and substrate specificity,HRV 3C protease is also used to remove fusion tags during protein purification process,presenting high industrial and medical values.Sequence analysis of viral polypeptide chains indicated that HRV 3C protease specifically recognized the LEVLFQ?GP sequence and cleaved between Gln and Gly.However,thorough characterization of the substrate specificity of HRV 3C protease has not been performed yet.In our studies here,we developed a new method based on the Yeast Endoplasmic reticulum Sequestration Screening(YESS)system,annotated as YESS-PSSC(YESS-Protease Substrate Specificity Characterization)that could rapidly resolve the substrate specificity of HRV 3C protease.Using the YESS-PSSC method,the amino acid specificities at the P1(Q),P1'(G),and P2'(P)positions were fully characterized.Comparing to the conventional in vitro protease-substrate method,the YESS-PSSC approach presented the advantages of avoiding the protein expression,purification,and in vitro reaction conditions optimization steps.Additionally,it could decipher the substrate specificity of protease in a simple,sensitive,and accurate way under the help of yeast ER retention sequence(ERS).We compare d the YESS-PSSC approach with the conventional in vitro protein substrate cleavage method by resolving the amino acid specificity of the less specific P2' position of HRV 3C protease.The results showed that the overall trend of the specific cutting efficiency for the 20 different amino acids at P2' position determined by these two methods was largely consistent with subtle difference,proving that the YESS-PSSC approach could effectively replace the conventional in vitro protein substrate cleavage method.Our comprehensive analysis of the substrate specificity of HRV 3C protease at P1(Q),P1'(G),and P2'(P)positions indicated that HRV 3C protease could efficiently recognize Gln,the original residue at the P1 position,as well as Gln,which both presented the substrate cutting efficiency over 96%.Additionally,HRV 3C protease could only recognize amino acids with small side chains at the P1' position.The substrate cutting efficiency was determined as 98%,71%,76%,and 35%,when the P1' residue was Gly,Ala,Cys and Ser,respectively.Moreover,our results also indicated the weak substrate specificity at the P2' position.Subsequently,we performed the structural simulation to dock the HRV 3C protease with various substrates.Combined with biochemical analysis,our results suggested that the specific recognition of residues at the P1 position were mainly determined by their interaction with His160 and Thr141 in HRV 3C protease.The saturation mutagenesis analysis agaisnt His160 and Thr141 showed that the hydrogen bond formed between the His160 of HRV 3C protease with the amino acid at the P1 position of the substrate was the main force for immobilizing the residue in the S1 binding pocket.In addition,two hydrogen bonds formed between Thr141 and the amino acid at the P1 position to stabilize the orientation of the substrate and maintain the structure of the S1 binding pocket.Instead of any hydrogen bonds,the small S1' binding pocket of HRV 3C protease determined that it could only accommodate amino acids with small side chains,thus having the highest recognition efficiency for Gly.Interestingly,in our analysis of the P2' position,it was found that HRV 3C protease preferred the Met residue over the original Pro residue,especially at 30 ?.When the in vitro proteolytic reactions were carried out at 4 ?,16 ?,22 ?,and 30 ? for 1 hour,respectively,the cleavage efficiency of HRV 3C protease against its original substrate LEVLFQ?GP decreased from 83.2% to 47.1% along with the increased temperature.In contrast,the cleavage efficiency against the substrate sequence LEVLFQ?GM was maintained at a high level,only decreased from 85.9% to 81.5%.We further analyzed the structures of HRV 3C protease at 6 ? and 30 ? using CD(circular dichroism)spectroscopy,and found that the increased temperature could cause the conversion of the random coils in HRV 3C protease to ?-turns.This conversion might cause the structural inflexibility of HRV 3C protease,thus resulting in the reduced cleavage efficiency of HRV 3C protease against the LEVLFQ?GP substrate.In addition,we also compared our results of HRV 3C protease with other typical viral 3C proteases,including foot-and-mouth disease virus 3C protease and tobacco mosaic virus protease.It was speculated that the neutral and acidic amino acids surrounding the S1 binding pocket determined the high specificity at the P1 position for Gln and Glu,while the small S1 ' binding pocket space might be the major distinguishing feature of HRV 3C protease.In summary,our research has developed a simple and easy in vivo method,YESS-PSSC,for analyzing the substrate specificity of protease.Using YESS-PSSC method,the substrate specificity of HRV 3C protease and its catalytic mechanism was further explored.Our studies laid a solid foundation for the future research,application,and molecular engineering of HRV 3C protease.