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PDBsum entry 2hob
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Viral protein
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PDB id
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2hob
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References listed in PDB file
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Key reference
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Title
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Production of authentic sars-Cov m(pro) with enhanced activity: application as a novel tag-Cleavage endopeptidase for protein overproduction.
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Authors
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X.Xue,
H.Yang,
W.Shen,
Q.Zhao,
J.Li,
K.Yang,
C.Chen,
Y.Jin,
M.Bartlam,
Z.Rao.
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Ref.
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J Mol Biol, 2007,
366,
965-975.
[DOI no: ]
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PubMed id
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Abstract
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The viral proteases have proven to be the most selective and useful for removing
the fusion tags in fusion protein expression systems. As a key enzyme in the
viral life-cycle, the main protease (M(pro)) is most attractive for drug design
targeting the SARS coronavirus (SARS-CoV), the etiological agent responsible for
the outbreak of severe acute respiratory syndrome (SARS) in 2003. In this study,
SARS-CoV M(pro) was used to specifically remove the GST tag in a new fusion
protein expression system. We report a new method to produce wild-type (WT)
SARS-CoV M(pro) with authentic N and C termini, and compare the activity of WT
protease with those of three different types of SARS-CoV M(pro) with additional
residues at the N or C terminus. Our results show that additional residues at
the N terminus, but not at the C terminus, of M(pro) are detrimental to enzyme
activity. To explain this, the crystal structures of WT SARS-CoV M(pro) and its
complex with a Michael acceptor inhibitor were determined to 1.6 Angstroms and
1.95 Angstroms resolution respectively. These crystal structures reveal that the
first residue of this protease is important for sustaining the substrate-binding
pocket and inhibitor binding. This study suggests that SARS-CoV M(pro) could
serve as a new tag-cleavage endopeptidase for protein overproduction, and the WT
SARS-CoV M(pro) is more appropriate for mechanistic characterization and
inhibitor design.
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Figure 3.
Figure 3. Superposition of the S1 pockets of GPLGS-WT and WT
SARS-CoV M^pro (in stereo). (a) Superposition of the S1 pockets
in protomer A of GPLGS-WT and that of protomer A* of WT SARS-CoV
M^pro. Protomer A* of WT is in blue; protomer A of GPLGS-WT is
in yellow; protomer B* of WT is in magenta; protomer B of
GPLGS-WT is in red. In the WT structure, the amino group (NH[2])
of Ser1 in protomer B* donates a 3.0 Å hydrogen bond to
the carboxylate group of Glu166 and a 2.7 Å hydrogen bond
to the main-chain carbonyl group of Phe140 in protomer A*,
stabilizing the S1 pocket. The NH of Gly143 moves 0.8 Å
toward the activity site; the main chain of residues 142-143
moves toward the S1 subsite; the side-chain of Asn-A*142 flips
over with a 6 Å shift compared with protomer A of
GPLGS-WT. (b) Superposition of the S1 pockets in protomer B of
GPLGS-WT and that of Protomer A* of WT SARS-CoV M^pro. Protomer
A* of WT is in blue; protomer B of GPLGS-WT is in yellow;
protomer B* of WT is in magenta; protomer A of GPLGS-WT is in
red. The S1 pocket of protomer B collapses partly with
reorientation of Glu166 and residues 140–143. No electron
density was visible for residues A1 and A2. Figure 3.
Superposition of the S1 pockets of GPLGS-WT and WT SARS-CoV
M^pro (in stereo). (a) Superposition of the S1 pockets in
protomer A of GPLGS-WT and that of protomer A* of WT SARS-CoV
M^pro. Protomer A* of WT is in blue; protomer A of GPLGS-WT is
in yellow; protomer B* of WT is in magenta; protomer B of
GPLGS-WT is in red. In the WT structure, the amino group (NH[2])
of Ser1 in protomer B* donates a 3.0 Å hydrogen bond to
the carboxylate group of Glu166 and a 2.7 Å hydrogen bond
to the main-chain carbonyl group of Phe140 in protomer A*,
stabilizing the S1 pocket. The NH of Gly143 moves 0.8 Å
toward the activity site; the main chain of residues 142-143
moves toward the S1 subsite; the side-chain of Asn-A*142 flips
over with a 6 Å shift compared with protomer A of
GPLGS-WT. (b) Superposition of the S1 pockets in protomer B of
GPLGS-WT and that of Protomer A* of WT SARS-CoV M^pro. Protomer
A* of WT is in blue; protomer B of GPLGS-WT is in yellow;
protomer B* of WT is in magenta; protomer A of GPLGS-WT is in
red. The S1 pocket of protomer B collapses partly with
reorientation of Glu166 and residues 140–143. No electron
density was visible for residues A1 and A2.
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Figure 4.
Figure 4. Differences between the complex structures of WT and
GPLGS-WT. (a) Inhibitor N3. (b) Superposition of the
substrate-binding pockets in protomer A of GPLGS-WT and that in
protomer A* of WT. In the WT-N3 complex structure, the NH[2]
group of Ser1 in protomer B* was still hydrogen-bonded to the
carboxylate group of Glu166 and the carbonyl group of Phe140 in
protomer A*, stabilizing the S1 pocket. In the GPLGS-WT-N3
complex structure, however, the two hydrogen bonds described
above were not found. Instead, an ordered water molecule was
observed in the S1 pocket. Protomer A* of WT is in blue;
protomer A of GPLGS-WT is in yellow; inhibitor N3 (complexed
with WT) is in magenta; inhibitor N3 (complexed with GPLGS-WT)
is in red; protomer B* of WT is in green; protomer B of GPLGS-WT
is in cyan. Figure 4. Differences between the complex
structures of WT and GPLGS-WT. (a) Inhibitor N3. (b)
Superposition of the substrate-binding pockets in protomer A of
GPLGS-WT and that in protomer A* of WT. In the WT-N3 complex
structure, the NH[2] group of Ser1 in protomer B* was still
hydrogen-bonded to the carboxylate group of Glu166 and the
carbonyl group of Phe140 in protomer A*, stabilizing the S1
pocket. In the GPLGS-WT-N3 complex structure, however, the two
hydrogen bonds described above were not found. Instead, an
ordered water molecule was observed in the S1 pocket. Protomer
A* of WT is in blue; protomer A of GPLGS-WT is in yellow;
inhibitor N3 (complexed with WT) is in magenta; inhibitor N3
(complexed with GPLGS-WT) is in red; protomer B* of WT is in
green; protomer B of GPLGS-WT is in cyan.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2007,
366,
965-975)
copyright 2007.
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