Critical assessment of important regions in the subunit association and catalytic action of the severe acute respiratory syndrome coronavirus main protease.
The severe acute respiratory syndrome (SARS) coronavirus (CoV) main protease represents an attractive target for the development of novel anti-SARS agents. The tertiary structure of the protease consists of two distinct folds. One is the N-terminal chymotrypsin-like fold that consists of two structural domains and constitutes the catalytic machinery; the other is the C-terminal helical domain, which has an unclear function and is not found in other RNA virus main proteases. To understand the functional roles of the two structural parts of the SARS-CoV main protease, we generated the full-length of this enzyme as well as several terminally truncated forms, different from each other only by the number of amino acid residues at the C- or N-terminal regions. The quaternary structure and K(d) value of the protease were analyzed by analytical ultracentrifugation. The results showed that the N-terminal 1-3 amino acid-truncated protease maintains 76% of enzyme activity and that the major form is a dimer, as in the wild type. However, the amino acids 1-4-truncated protease showed the major form to be a monomer and had little enzyme activity. As a result, the fourth amino acid seemed to have a powerful effect on the quaternary structure and activity of this protease. The last C-terminal helically truncated protease also exhibited a greater tendency to form monomer and showed little activity. We concluded that both the C- and the N-terminal regions influence the dimerization and enzyme activity of the SARS-CoV main protease.
Hsu
Wen-Chi
WC
Faculty of Life Sciences, Institute of Biochemistry, and Structural Biology Program, National Yang-Ming University, Taipei 112, Taiwan.
Chang
Hui-Chuan
HC
Chou
Chi-Yuan
CY
Tsai
Pui-Jen
PJ
Lin
Pei-In
PI
Chang
Gu-Gang
GG
D002384:Catalysis; D002918:Chymotrypsin / Q000737:chemistry; D002942:Circular Dichroism; D000086782:Coronavirus 3C Proteases; D018360:Crystallography, X-Ray; D003546:Cysteine Endopeptidases; D019281:Dimerization; D010450:Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D017353:Gene Deletion; D007700:Kinetics; D008958:Models, Molecular; D015233:Models, Statistical; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000737:chemistry; D045473:Severe acute respiratory syndrome-related coronavirus / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D014461:Ultracentrifugation; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism
D012367:RNA, Viral; D014764:Viral Proteins; D010450:Endopeptidases; D002918:Chymotrypsin; D003546:Cysteine Endopeptidases; D000086782:Coronavirus 3C Proteases