title: Autoprocessing mechanism of severe acute respiratory syndrome coronavirus 3C-like protease (SARS-CoV 3CLpro) from its polyproteins.
abstract: Like many other RNA viruses, severe acute respiratory syndrome coronavirus (SARS-CoV) produces polyproteins containing several non-structural proteins, which are then processed by the viral proteases. These proteases often exist within the polyproteins, and are excised by their own proteolytic activity ('autoprocessing'). It is important to investigate the autoprocessing mechanism of these proteases from the point of view of anti-SARS-CoV drug design. In this paper, we describe a new method for investigating the autoprocessing mechanism of the main protease (M(pro)), which is also called the 3C-like protease (3CL(pro)). Using our method, we measured the activities, under the same conditions, of the mature form and pro-forms with the N-terminal pro-sequence, the C-terminal pro-sequence or both pro-sequences, toward the pro-form with both N- and C-terminal pro-sequences. The data indicate that the pro-forms of the enzyme have proteolytic activity, and are stimulated by the same proteolytic activity. The stimulation occurs in two steps, with approximately eightfold stimulation by N-terminal cleavage, approximately fourfold stimulation by C-terminal cleavage, and 23-fold stimulation by the cleavage of both termini, compared to the pro-form with both the N- and C-terminal pro-sequences. Such cleavage mainly occurs in a trans manner; i.e. the pro-form dimer cleaves the monomeric form. The stimulation by N-terminal pro-sequence removal is due to the cis (intra-dimer and inter-protomer) effect of formation of the new N-terminus, whereas that by C-terminal cleavage is due to removal of its trans (inter-dimer) inhibitory effect. A numerical simulation of the maturation pathway is presented.
authors: RIKEN Systems and Structural Biology Center, Tsurumi, Yokohama, Japan. tmuramat@gsc.riken.jp
authors: Tomonari
authors: T
authors: Muramatsu
authors: Yong-Tae
authors: YT
authors: Kim
authors: Wataru
authors: W
authors: Nishii
authors: Takaho
authors: T
authors: Terada
authors: Mikako
authors: M
authors: Shirouzu
authors: Shigeyuki
authors: S
authors: Yokoyama
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000086782:Coronavirus 3C Proteases; D003546:Cysteine Endopeptidases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D057075:Enzyme Assays; D004926:Escherichia coli; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000737:chemistry; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D020815:Polyproteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D045473:Severe acute respiratory syndrome-related coronavirus / Q000201:enzymology*; D014764:Viral Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics
chemical_list: D020815:Polyproteins; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins; D003546:Cysteine Endopeptidases; D000086782:Coronavirus 3C Proteases