Mechanism of the Maturation Process of SARS-CoV 3CL Protease
Resource
Journal of Biological Chemistry 280 (35): 31257-31266
Journal
Journal of Biological Chemistry
Pages
31257-31266
Date Issued
2005
Date
2005
Author(s)
Hsu, Min-Feng
Kuo, Chih-Jung
Chang, Kai-Ti
Chang, Hui-Chuan
Chou, Chia-Cheng
Ko, Tzu-Ping
Shr, Hui-Lin
Chang, Gu-Gang
Wang, Andrew H.-J.
Liang, Po-Huang
Abstract
Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by a novel human coronavirus. Viral maturation requires a main protease (3CLpro) to cleave the virus-encoded polyproteins. We report here that the 3CLpro containing additional N- and/or C-terminal segments of the polyprotein sequences undergoes autoprocessing and yields the mature protease in vitro. The dimeric three-dimensional structure of the C145A mutant protease shows that the active site of one protomer binds with the C-terminal six amino acids of the protomer from another asymmetric unit, mimicking the product-bound form and suggesting a possible mechanism for maturation. The P1 pocket of the active site binds the Gln side chain specifically, and the P2 and P4 sites are clustered together to accommodate large hydrophobia side chains. The tagged C145A mutant protein served as a substrate for the wild-type protease, and the N terminus was first digested (55-fold faster) at the Gln-1-Ser1 site followed by the C-terminal cleavage at the Gln306-Gly307 site. Analytical ultracentrifuge of the quaternary structures of the tagged and mature proteases reveals the remarkably tighter dimer formation for the mature enzyme (Kd = 0.35 nM) than for the mutant (C145A) containing 10 extra N-terminal (Kd = 17.2 nM) or C-terminal amino acids (Kd = 5.6 nM). The data indicate that immature 3CLpro can form dimer enabling it to undergo autoprocessing to yield the mature enzyme, which further serves as a seed for facilitated maturation. Taken together, this study provides insights into the maturation process of the SARS 3CLpro from the polyprotein and design of new structure-based inhibitors. ? 2005 by The American Society for Biochemistry and Molecular Biology, Inc.
SDGs
Other Subjects
Amino acids; Crystal structure; Diseases; Molecular biology; C-terminal segments; Mimicking; Severe acute respiratory syndrome (SARS); Viral maturation; Enzymes; glutamine; glycine; polyprotein; proteinase; article; carboxy terminal sequence; cell maturation; enzyme active site; enzyme mechanism; enzyme substrate; hydrophobicity; priority journal; protein processing; SARS coronavirus; severe acute respiratory syndrome; ultracentrifugation; virogenesis; Binding Sites; Crystallography, X-Ray; Cysteine Endopeptidases; Endopeptidases; Humans; Models, Molecular; Molecular Structure; Protein Processing, Post-Translational; Protein Structure, Tertiary; Recombinant Fusion Proteins; SARS Virus; Viral Proteins; human coronavirus; SARS CoV
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