Biosynthesis, purification, and substrate specificity of severe acute respiratory syndrome coronavirus 3C-like proteinase

被引:268
作者
Fan, KQ
Wei, P
Feng, Q
Chen, SD
Huang, CK
Ma, L
Lai, B
Pei, JF
Liu, Y
Chen, JG
Lai, LH [1 ]
机构
[1] Peking Univ, Coll Chem, State Key Lab Struct Chem Stable & Unstable Speci, Beijing 100871, Peoples R China
[2] Peking Univ, Coll Life Sci, Dept Cell Biol & Genet, Beijing 100871, Peoples R China
[3] Peking Univ, Ctr Theoret Biol, Beijing 100871, Peoples R China
关键词
D O I
10.1074/jbc.M310875200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The 3C-like proteinase of severe acute respiratory syndrome (SARS) coronavirus has been proposed to be a key target for structural-based drug design against SARS. In order to understand the active form and the substrate specificity of the enzyme, we have cloned, expressed, and purified SARS 3C-like proteinase. Analytic gel filtration shows a mixture of monomer and dimer at a protein concentration of 4 mg/ml and mostly monomer at 0.2 mg/ml, which correspond to the concentration used in the enzyme assays. The linear decrease of the enzymatic-specific activity with the decrease of enzyme concentration revealed that only the dimeric form is active and the dimeric interface could be targeted for structural-based drug design against SARS 3C-like proteinase. By using a high pressure liquid chromatography assay, SARS 3C-like proteinase was shown to cut the 11 peptides covering all of the 11 cleavage sites on the viral polyprotein with different efficiency. The two peptides corresponding to the two self-cleavage sites are the two with highest cleavage efficiency, whereas peptides with non-canonical residues at P2 or P1' positions react slower. The P2 position of the substrates seems to favor large hydrophobic residues. Secondary structure studies for the peptide substrates revealed that substrates with more beta-sheetlike structure tend to react fast. This study provides a basic understanding of the enzyme catalysis and a full substrate specificity spectrum for SARS 3C-like proteinase, which are helpful for structural-based inhibitor design against SARS and other coronavirus.
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页码:1637 / 1642
页数:6
相关论文
共 24 条
  • [1] Coronavirus main proteinase (3CLpro) structure:: Basis for design of anti-SARS drugs
    Anand, K
    Ziebuhr, J
    Wadhwani, P
    Mesters, JR
    Hilgenfeld, R
    [J]. SCIENCE, 2003, 300 (5626) : 1763 - 1767
  • [2] Structure of coronavirus main proteinase reveals combination of a chymotrypsin fold with an extra α-helical domain
    Anand, K
    Palm, GJ
    Mesters, JR
    Siddell, SG
    Ziebuhr, J
    Hilgenfeld, R
    [J]. EMBO JOURNAL, 2002, 21 (13) : 3213 - 3224
  • [3] Binding mechanism of coronavirus main proteinase with ligands and its implication to drug design against SARS
    Chou, KC
    Wei, DQ
    Zhong, WZ
    [J]. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 2003, 308 (01) : 148 - 151
  • [4] CORDINGLEY MG, 1990, J BIOL CHEM, V265, P9062
  • [5] Structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3C protease inhibitors. Part 7: Structure-activity studies of bicyclic 2-pyridone-containing peptidomimetics
    Dragovich, PS
    Prins, TJ
    Zhou, R
    Johnson, TO
    Brown, EL
    Maldonado, FC
    Fuhrman, SA
    Zalman, LS
    Patick, AK
    Matthews, DA
    Hou, XJ
    Meador, JW
    Ferre, RA
    Worland, ST
    [J]. BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 2002, 12 (05) : 733 - 738
  • [6] Structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3C protease inhibitors. 6. Structure-activity studies of orally bioavailable, 2-pyridone-containing peptidomimetics
    Dragovich, PS
    Prins, TJ
    Zhou, R
    Brown, EL
    Maldonado, FC
    Fuhrman, SA
    Zalman, LS
    Tuntland, T
    Lee, CA
    Patick, AK
    Matthews, DA
    Hendrickson, TF
    Kosa, MB
    Liu, B
    Batugo, MR
    Gleeson, JPR
    Sakata, SK
    Chen, LJ
    Guzman, MC
    Meador, JW
    Ferre, RA
    Worland, ST
    [J]. JOURNAL OF MEDICINAL CHEMISTRY, 2002, 45 (08) : 1607 - 1623
  • [7] Structure-based design of ketone-containing, tripeptidyl human rhinovirus 3C protease inhibitors
    Dragovich, PS
    Zhou, R
    Webber, SE
    Prins, TJ
    Kwok, AK
    Okano, K
    Fuhrman, SA
    Zalman, LS
    Maldonado, FC
    Brown, EL
    Meador, JW
    Patick, AK
    Ford, CE
    Brothers, MA
    Binford, SL
    Matthews, DA
    Ferre, RA
    Worland, ST
    [J]. BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 2000, 10 (01) : 45 - 48
  • [8] Structure-based design of irreversible, tripeptidyl human rhinovirus 3C protease inhibitors containing N-methyl amino acids
    Dragovich, PS
    Webber, SE
    Prins, TJ
    Zhou, R
    Marakovits, JT
    Tikhe, JG
    Fuhrman, SA
    Patick, AK
    Matthews, DA
    Ford, CE
    Brown, EL
    Binford, SL
    Meador, JW
    Ferre, RA
    Worland, ST
    [J]. BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 1999, 9 (15) : 2189 - 2194
  • [9] Structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3C protease inhibitors.: 4.: Incorporation of P1 lactam moieties as L-glutamine replacements
    Dragovich, PS
    Prins, TJ
    Zhou, R
    Webber, SE
    Marakovits, JT
    Fuhrman, SA
    Patick, AK
    Matthews, DA
    Lee, CA
    Ford, CE
    Burke, BJ
    Rejto, PA
    Hendrickson, TF
    Tuntland, T
    Brown, EL
    Meador, JW
    Ferre, RA
    Harr, JEV
    Kosa, MB
    Worland, ST
    [J]. JOURNAL OF MEDICINAL CHEMISTRY, 1999, 42 (07) : 1213 - 1224
  • [10] Structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3C protease inhibitors. 3. Structure - Activity studies of ketomethylene-containing peptidomimetics
    Dragovich, PS
    Prins, TJ
    Zhou, R
    Fuhrman, SA
    Patick, AK
    Matthews, DA
    Ford, CE
    Meador, JW
    Ferre, RA
    Worland, ST
    [J]. JOURNAL OF MEDICINAL CHEMISTRY, 1999, 42 (07) : 1203 - 1212