| African swine fever virus(ASFV)is the only member of Asfivirus genus in the Asfarviridae family,which can infect domestic and wild pigs with highly infectious and mortality.ASFV has a unique five-layer structure.The genome-containing nucleoid is surrounded by a thick protein layer referred to as the core shell,and an inner lipid envelope,and wrapped by a protein capsid,forming an icosahedral structure.Extracellular ASFV gains an external envelope(the fifth layer)as it buds through the plasma membrane,packaged into virus particles with a diameter of about 260 nm.This unique five layer structure is very rare among viruses,which indicates the complexity of virus particle assembly and the complexity of host mechanism of ASFV infection,with a genome size of 170-190 kbp,and composed of151-167 open reading frames.NP868 R gene has been proved to be related to the post transcription modification of virus mRNA..pNP868 R is a capping enzyme of ASFV.It has 868 amino acids which is composed of three domains: triphosphatase(TPase)domain at N-terminal,guanylltransferase(GTase)domain and methyltransferase(MTase)domain at C-terminal,MTase transfers the methylation of S-adenosylmethionine to the N7 position of guanine,form a cap0 structure.Although the activities of these three enzymes have been well known,the mechanism and structure of p NP868 R catalyzing mRNA capping have not been reported.Based on the recombinant expression and purification of methyltransferase domain(MT),we carried out the co-crystallization experiment of MT and methy donor s-adenosinemethionine(AdoMet),and obtained high-quality protein crystal.High resolution diffraction data were collected at SSRF,and the crystal structure of the complex with resolution of 2.7 ?was successfully resolved by molecular displacement method.The results showed that MT exhibited ca-nonical class ? MTase family fold comprising a seven-strand b-sheet with three helices on each side.In addition,different from the N-terminal flexible conformation of methyltransferase in most cells,the N-terminal of the complex structure is ordered.,and far away from the the AdoMet binding site,however,in the D1 subunit of poxvirus capping enzyme,the N-terminal is closely over the active site.AdoMet is bound in a negative charge pocket,and linked with the surrounding residues through hydrogen bond,ionic bondand and hydrophobic interaction.Through the study of the site mutation of these residues and ITC experiments,find the K607,N709,F711 and G624 play an important role in the identification and binding of AdoMet.The cap analog(m7Gppp G)from eukaryotic mRNA capping enzyme can be docked into the putative substrate binding pocket with predominantly positive charges without steric clash by their superposition and the residue which might participate in the activity of methyltransferase was found.K607,R633 and R658 were identified as key sites by enzyme activity analysis,and is also found that most of the binding sites were highly conserved in the homologous structure.The N-terminal extension may play an important role in substrate binding.In addition,we also analyzed the surface potential of MT structure and homologous structure,and found that there is a surface path formed by positive charge near their substrate binding active sites.Combined with related studies,we analyzed that this may provide a pathway for binding to substrate RNA.Our structural and biochemical studies of methyltransferase MT provide new insights into the process of methyltransferase in capping reaction,enrich the understanding of the structural mechanism of the catalytic cap of p NP868 R and provide a basis for the design of potential anti ASFV inhibitors targeting key enzymes. |
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