Molecular Mechanism For Coronavirus PEDV 3CL^pro Substrate Recognition And SARS-CoV-2 Receptor Recognition

Coronaviruses,widely spread in nature,can infect humans and many species of animals,mainly causing the respiratory tract and intestines related disease.Some of the diseases are extremely contagious or have a high lethal rate,thus are important pathogens related to animal husbandry or human public health.3CL^pro,Rd Rp and S protein are three important anti-coronavirus targets.During coronavirus replication,3CL^pro is responsible for the cleavage and maturation of the majority of the non-structural proteins at the carboxy terminus of viral polyproteins,and Rd Rp is the core of the RTC.Both proteins have high homology and conservative catalytic mechanism in different genera of coronavirus,thus served as very promising antiviral targets.S protein is responsible for the coronavirus attachment to cell receptors and membrane fusion.It is the key factor for viral infection and is therefore a vital target for the development of vaccines and neutralizing antibodies.This study focuses on two important targets,3CL^pro from PEDV which is an important pathogen in the pig breeding industry,and S RBD from SARS-Co V-2 which currently spread throughout the world.The structural researches regarding the two targets here provide knowledge for the development of anti-coronavirus strategies.The study are as follows:1. The structural basis of PEDV 3CL^pro dimerization and peptide substrates recognition.We determined that PEDV 3CL^pro exists as a dimer in solution by using size exclusion chromatography and analytical ultracentrifugation.We solved the crystal structure of the wild-type PEDV 3CL^pro.Structure based mutagenesis analysis of the residues on the dimer interface reveal that PEDV 3CL^pro forms a stable dimer,and point mutation is unable to destroy its dimer form but will depress the enzymatic activity;however,the deletion of the N-terminal eight residues would destroy and inactivate the 3CL^pro dimer.In order to illustrate the structural basis of PEDV 3CL^pro substrate recognition,the crystal structure of PEDV 3CL^pro?C144A?-peptide complex was determined.PEDV 3CL^pro recognize Q at P1 position,prefer residues like L and M that has large hydrophobic side chains at P2 position and prefer small side chain residues such as S,G,and A at the P1'position.Further hydrolysis assays with three fluorescent peptide substrates indicate that PEDV 3CL^pro can tolerate amino acids with larger side chains,such as V and N at the S1'binding subsite,however SARS-Co V 3CL^pro can't.Meanwhile,some non-conserved motifs in the binding pockets of different coronavirus 3CL^pros may also affect the substrate recognition to a certain extent.2. The structural basis for inhibiting PEDV replication by GC376 targeting the 3CL^pro.We determined that GC376 effectively inhibited the 3CL^pro enzymatic activity and PEDV infection of host cells.To explain the molecular basis of GC376 inhibiting PEDV infection,we solved the crystal structure of the PEDV 3CL^pro-GC376 complex.Finally,we compared our PEDV 3CL^pro-GC376 structure with the structure of the TGEV 3CL^pro-GC376 complex.They showed similar binding mode but also some structural differences.3. The structural basis of SARS-Co V-2 receptor recognition.We designed the SARS-Co V-2 chimeric RBD and subsequently solved the crystal structure of the RBD-h ACE2complex.The structure clearly shows binding details of the RBD and its receptor.And a 4-residues motif?GVEG?of the RBM makes it closer to h ACE2,resulting in more contacts and a higher binding affinity than that between SARS-Co V RBD and h ACE2.Biochemical experiments further proved this conclusion.Compared to SARS-Co V RBD-h ACE2 structure,we found that viral binding hot spots 31 and 353 of h ACE2 have undergone subtle conformation changes.The salt bridge between K31 and h ACE2 E35 breaks apart,and each of the residues forms a hydrogen bond with Gln493 of the SARS-Co V-2 RBM.Viral binding hot residue K353 forms a new hydrogen bond with the main chain of G496 of the SARS-Co V-2RBM while maintaining the salt bridge with Asp38 of h ACE2.Both viral binding hot spots maintains its stability by adjusting the interactions between itself and surrounding residues both from h ACE2 and RBM,thus still plays an important role in viral binding.It reveals the ingenious changes of SARS-Co V-2 during evolution,and partly explains the high infectivity.And also provides structural basis for neutralizing antibody develop,optimization and vaccine design.In summary,our study focuses on two important Co Vs,PEDV and SARS-Co V-2.We explored the structural basis of PEDV 3CL^pro dimerization,substrate recognition and the inhibiting effect of GC376 against PEDV 3CL^pro,and then clarified the structural basis of SARS-Co V-2 receptor recognition of spike protein.We focused on the key proteins during coronavirus replication and cell invasion.This study provides structural basis for the development of anti-coronavirus drugs,vaccines or antibodies against different targets.