Essential covalent linkage between the chymotrypsin-like domain and the extra domain of the SARS-CoV main protease.
Identifieur interne : 002543 ( Main/Exploration ); précédent : 002542; suivant : 002544Essential covalent linkage between the chymotrypsin-like domain and the extra domain of the SARS-CoV main protease.
Auteurs : Meng-Ying Tsai [Taïwan] ; Wei-Hsin Chang ; Jin-Yi Liang ; Long-Liu Lin ; Gu-Gang Chang ; Hui-Ping ChangSource :
- Journal of biochemistry [ 1756-2651 ] ; 2010.
Descripteurs français
- KwdFr :
- MESH :
English descriptors
- KwdEn :
- MESH :
- chemical , chemistry : Cysteine Endopeptidases, Viral Proteins.
- chemical : Chymotrypsin, Protein Subunits.
- enzymology : SARS Virus.
- Protein Structure, Quaternary, Protein Structure, Tertiary.
Abstract
The main protease of the coronavirus causing severe acute respiratory syndrome performs proteolytic processing of the viral polyproteins. The active form of the enzyme is a homodimer with each subunit consisting of three structural domains. Domains I and II, hosting the complete catalytic machinery, constitute the N-terminal chymotrypsin-like folding scaffold and connect to the extra C-terminal domain III by a long loop. Previously, the domain III-truncated enzyme was demonstrated to fold independently into an intact chymotrypsin-like fold, but it showed no enzyme activity. To further delineate the structure-function relationships of the domain III and the long loop, we generated some truncated and mutated M(pro) forms bearing various combinations of the loop with other structural parts of the enzyme. Their conformational and association properties were investigated in detail. Far-ultraviolet circular dichroism (CD) measurements revealed that these fragments could fold independently. The secondary, tertiary and quaternary structures of these mixtures were monitored by CD, fluorescence spectroscopy and analytical ultracentrifugation. However, no enzyme activity was observed for any mutant or mixtures. These observations indicate that the covalent linkage between the chymotrypsin like and the extra domain is essential for enzymatic activity of the main coronavirus protease and for the integrity of its quaternary structure.
DOI: 10.1093/jb/mvq071
PubMed: 20587646
Affiliations:
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uniqKey="Liang J" first="Jin-Yi" last="Liang">Jin-Yi Liang</name></author><author><name sortKey="Lin, Long Liu" sort="Lin, Long Liu" uniqKey="Lin L" first="Long-Liu" last="Lin">Long-Liu Lin</name></author><author><name sortKey="Chang, Gu Gang" sort="Chang, Gu Gang" uniqKey="Chang G" first="Gu-Gang" last="Chang">Gu-Gang Chang</name></author><author><name sortKey="Chang, Hui Ping" sort="Chang, Hui Ping" uniqKey="Chang H" first="Hui-Ping" last="Chang">Hui-Ping Chang</name></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2010">2010</date><idno type="RBID">pubmed:20587646</idno><idno type="pmid">20587646</idno><idno type="doi">10.1093/jb/mvq071</idno><idno type="wicri:Area/PubMed/Corpus">001674</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Corpus" wicri:corpus="PubMed">001674</idno><idno type="wicri:Area/PubMed/Curation">001674</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Curation">001674</idno><idno 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xml:lang="fr">Taïwan</country><wicri:regionArea>Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, 155 Li-Nong St., Section 2, Taipei 112</wicri:regionArea><wicri:noRegion>Taipei 112</wicri:noRegion></affiliation></author><author><name sortKey="Chang, Wei Hsin" sort="Chang, Wei Hsin" uniqKey="Chang W" first="Wei-Hsin" last="Chang">Wei-Hsin Chang</name></author><author><name sortKey="Liang, Jin Yi" sort="Liang, Jin Yi" uniqKey="Liang J" first="Jin-Yi" last="Liang">Jin-Yi Liang</name></author><author><name sortKey="Lin, Long Liu" sort="Lin, Long Liu" uniqKey="Lin L" first="Long-Liu" last="Lin">Long-Liu Lin</name></author><author><name sortKey="Chang, Gu Gang" sort="Chang, Gu Gang" uniqKey="Chang G" first="Gu-Gang" last="Chang">Gu-Gang Chang</name></author><author><name sortKey="Chang, Hui Ping" sort="Chang, Hui Ping" uniqKey="Chang H" first="Hui-Ping" last="Chang">Hui-Ping Chang</name></author></analytic><series><title level="j">Journal of biochemistry</title><idno type="eISSN">1756-2651</idno><imprint><date when="2010" type="published">2010</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Chymotrypsin</term><term>Cysteine Endopeptidases (chemistry)</term><term>Protein Structure, Quaternary</term><term>Protein Structure, Tertiary</term><term>Protein Subunits</term><term>SARS Virus (enzymology)</term><term>Viral Proteins (chemistry)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Chymotrypsine</term><term>Cysteine endopeptidases ()</term><term>Protéines virales ()</term><term>Sous-unités de protéines</term><term>Structure quaternaire des protéines</term><term>Structure tertiaire des protéines</term><term>Virus du SRAS (enzymologie)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Cysteine Endopeptidases</term><term>Viral Proteins</term></keywords><keywords scheme="MESH" type="chemical" xml:lang="en"><term>Chymotrypsin</term><term>Protein Subunits</term></keywords><keywords scheme="MESH" qualifier="enzymologie" xml:lang="fr"><term>Virus du SRAS</term></keywords><keywords scheme="MESH" qualifier="enzymology" xml:lang="en"><term>SARS Virus</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Protein Structure, Quaternary</term><term>Protein Structure, Tertiary</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Chymotrypsine</term><term>Cysteine endopeptidases</term><term>Protéines virales</term><term>Sous-unités de protéines</term><term>Structure quaternaire des protéines</term><term>Structure tertiaire des protéines</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The main protease of the coronavirus causing severe acute respiratory syndrome performs proteolytic processing of the viral polyproteins. The active form of the enzyme is a homodimer with each subunit consisting of three structural domains. Domains I and II, hosting the complete catalytic machinery, constitute the N-terminal chymotrypsin-like folding scaffold and connect to the extra C-terminal domain III by a long loop. Previously, the domain III-truncated enzyme was demonstrated to fold independently into an intact chymotrypsin-like fold, but it showed no enzyme activity. To further delineate the structure-function relationships of the domain III and the long loop, we generated some truncated and mutated M(pro) forms bearing various combinations of the loop with other structural parts of the enzyme. Their conformational and association properties were investigated in detail. Far-ultraviolet circular dichroism (CD) measurements revealed that these fragments could fold independently. The secondary, tertiary and quaternary structures of these mixtures were monitored by CD, fluorescence spectroscopy and analytical ultracentrifugation. However, no enzyme activity was observed for any mutant or mixtures. These observations indicate that the covalent linkage between the chymotrypsin like and the extra domain is essential for enzymatic activity of the main coronavirus protease and for the integrity of its quaternary structure.</div></front></TEI><affiliations><list><country><li>Taïwan</li></country></list><tree><noCountry><name sortKey="Chang, Gu Gang" sort="Chang, Gu Gang" uniqKey="Chang G" first="Gu-Gang" last="Chang">Gu-Gang Chang</name><name sortKey="Chang, Hui Ping" sort="Chang, Hui Ping" uniqKey="Chang H" first="Hui-Ping" last="Chang">Hui-Ping Chang</name><name sortKey="Chang, Wei Hsin" sort="Chang, Wei Hsin" uniqKey="Chang W" first="Wei-Hsin" last="Chang">Wei-Hsin Chang</name><name sortKey="Liang, Jin Yi" sort="Liang, Jin Yi" uniqKey="Liang J" first="Jin-Yi" last="Liang">Jin-Yi Liang</name><name sortKey="Lin, Long Liu" sort="Lin, Long Liu" uniqKey="Lin L" first="Long-Liu" last="Lin">Long-Liu Lin</name></noCountry><country name="Taïwan"><noRegion><name sortKey="Tsai, Meng Ying" sort="Tsai, Meng Ying" uniqKey="Tsai M" first="Meng-Ying" last="Tsai">Meng-Ying Tsai</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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