Severe acute respiratory syndrome coronavirus 3C-like proteinase N terminus is indispensable for proteolytic activity but not for enzyme dimerization. Biochemical and thermodynamic investigation in conjunction with molecular dynamics simulations.
Identifieur interne : 004633 ( Main/Exploration ); précédent : 004632; suivant : 004634Severe acute respiratory syndrome coronavirus 3C-like proteinase N terminus is indispensable for proteolytic activity but not for enzyme dimerization. Biochemical and thermodynamic investigation in conjunction with molecular dynamics simulations.
Auteurs : Shuai Chen [République populaire de Chine] ; Lili Chen ; Jinzhi Tan ; Jing Chen ; Li Du ; Tao Sun ; Jianhua Shen ; Kaixian Chen ; Hualiang Jiang ; Xu ShenSource :
- The Journal of biological chemistry [ 0021-9258 ] ; 2005.
Descripteurs français
- KwdFr :
- Calorimétrie, Conformation des protéines, Cysteine endopeptidases, Dimérisation, Endopeptidases (), Endopeptidases (métabolisme), Protéines virales (), Protéines virales (métabolisme), Régions promotrices (génétique), Sites de fixation, Spécificité du substrat, Thermodynamique, Transfert d'énergie, Virus du SRAS (enzymologie).
- MESH :
- enzymologie : Virus du SRAS.
- métabolisme : Endopeptidases, Protéines virales.
- Calorimétrie, Conformation des protéines, Cysteine endopeptidases, Dimérisation, Endopeptidases, Protéines virales, Régions promotrices (génétique), Sites de fixation, Spécificité du substrat, Thermodynamique, Transfert d'énergie.
English descriptors
- KwdEn :
- Binding Sites, Calorimetry, Cysteine Endopeptidases, Dimerization, Endopeptidases (chemistry), Endopeptidases (metabolism), Energy Transfer, Promoter Regions, Genetic, Protein Conformation, SARS Virus (enzymology), Substrate Specificity, Thermodynamics, Viral Proteins (chemistry), Viral Proteins (metabolism).
- MESH :
- chemical , chemistry : Endopeptidases, Viral Proteins.
- chemical , metabolism : Endopeptidases, Viral Proteins.
- chemical : Cysteine Endopeptidases.
- enzymology : SARS Virus.
- Binding Sites, Calorimetry, Dimerization, Energy Transfer, Promoter Regions, Genetic, Protein Conformation, Substrate Specificity, Thermodynamics.
Abstract
Severe acute respiratory syndrome (SARS) coronavirus is a novel human coronavirus and is responsible for SARS infection. SARS coronavirus 3C-like proteinase (SARS 3CL(pro)) plays key roles in viral replication and transcription and is an attractive target for anti-SARS drug discovery. In this report, we quantitatively characterized the dimerization features of the full-length and N-terminal residues 1-7 deleted SARS 3CL(pro)s by using glutaraldehyde cross-linking SDS-PAGE, size-exclusion chromatography, and isothermal titration calorimeter techniques. Glutaraldehyde cross-linking SDS-PAGE and size-exclusion chromatography results show that, similar to the full-length SARS 3CL(pro), the N-terminal deleted SARS 3CL(pro) still remains a dimer/monomer mixture within a wide range of protein concentrations. Isothermal titration calorimeter determinations indicate that the equilibrium dissociation constant (K(d)) of the N-terminal deleted proteinase dimer (262 microm) is very similar to that of the full-length proteinase dimer (227 microm). Enzymatic activity assay using the fluorescence resonance energy transfer method reveals that N-terminal deletion results in almost complete loss of enzymatic activity for SARS 3CL(pro). Molecular dynamics and docking simulations demonstrate the N-terminal deleted proteinase dimer adopts a state different from that of the full-length proteinase dimer, which increases the angle between the two protomers and reduces the binding pocket that is not beneficial to the substrate binding. This conclusion is verified by the surface plasmon resonance biosensor determination, indicating that the model substrate cannot bind to the N-terminal deleted proteinase. These results suggest the N terminus is not indispensable for the proteinase dimerization but may fix the dimer at the active state and is therefore vital to enzymatic activity.
DOI: 10.1074/jbc.M408211200
PubMed: 15507456
Affiliations:
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Biochemical and thermodynamic investigation in conjunction with molecular dynamics simulations.</title><author><name sortKey="Chen, Shuai" sort="Chen, Shuai" uniqKey="Chen S" first="Shuai" last="Chen">Shuai Chen</name><affiliation wicri:level="1"><nlm:affiliation>Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Graduate School of Chinese Academy of Sciences, Shanghai 201203, China.</nlm:affiliation><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Graduate School of Chinese Academy of Sciences, Shanghai 201203</wicri:regionArea><wicri:noRegion>Shanghai 201203</wicri:noRegion></affiliation></author><author><name sortKey="Chen, Lili" sort="Chen, Lili" uniqKey="Chen L" first="Lili" last="Chen">Lili Chen</name></author><author><name sortKey="Tan, Jinzhi" sort="Tan, Jinzhi" uniqKey="Tan J" first="Jinzhi" last="Tan">Jinzhi Tan</name></author><author><name sortKey="Chen, Jing" sort="Chen, Jing" uniqKey="Chen J" first="Jing" last="Chen">Jing Chen</name></author><author><name sortKey="Du, Li" sort="Du, Li" uniqKey="Du L" first="Li" last="Du">Li Du</name></author><author><name sortKey="Sun, Tao" sort="Sun, Tao" uniqKey="Sun T" first="Tao" last="Sun">Tao Sun</name></author><author><name sortKey="Shen, Jianhua" sort="Shen, Jianhua" uniqKey="Shen J" first="Jianhua" last="Shen">Jianhua Shen</name></author><author><name sortKey="Chen, Kaixian" sort="Chen, Kaixian" uniqKey="Chen K" first="Kaixian" last="Chen">Kaixian Chen</name></author><author><name sortKey="Jiang, Hualiang" sort="Jiang, Hualiang" uniqKey="Jiang H" first="Hualiang" last="Jiang">Hualiang Jiang</name></author><author><name sortKey="Shen, Xu" sort="Shen, Xu" uniqKey="Shen X" first="Xu" last="Shen">Xu Shen</name></author></analytic><series><title level="j">The Journal of biological chemistry</title><idno type="ISSN">0021-9258</idno><imprint><date when="2005" type="published">2005</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Binding Sites</term><term>Calorimetry</term><term>Cysteine Endopeptidases</term><term>Dimerization</term><term>Endopeptidases (chemistry)</term><term>Endopeptidases (metabolism)</term><term>Energy Transfer</term><term>Promoter Regions, Genetic</term><term>Protein Conformation</term><term>SARS Virus (enzymology)</term><term>Substrate Specificity</term><term>Thermodynamics</term><term>Viral Proteins (chemistry)</term><term>Viral Proteins (metabolism)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Calorimétrie</term><term>Conformation des protéines</term><term>Cysteine endopeptidases</term><term>Dimérisation</term><term>Endopeptidases ()</term><term>Endopeptidases (métabolisme)</term><term>Protéines virales ()</term><term>Protéines virales (métabolisme)</term><term>Régions promotrices (génétique)</term><term>Sites de fixation</term><term>Spécificité du substrat</term><term>Thermodynamique</term><term>Transfert d'énergie</term><term>Virus du SRAS (enzymologie)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Endopeptidases</term><term>Viral Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Endopeptidases</term><term>Viral Proteins</term></keywords><keywords scheme="MESH" type="chemical" xml:lang="en"><term>Cysteine Endopeptidases</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" qualifier="métabolisme" xml:lang="fr"><term>Endopeptidases</term><term>Protéines virales</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Binding Sites</term><term>Calorimetry</term><term>Dimerization</term><term>Energy Transfer</term><term>Promoter Regions, Genetic</term><term>Protein Conformation</term><term>Substrate Specificity</term><term>Thermodynamics</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Calorimétrie</term><term>Conformation des protéines</term><term>Cysteine endopeptidases</term><term>Dimérisation</term><term>Endopeptidases</term><term>Protéines virales</term><term>Régions promotrices (génétique)</term><term>Sites de fixation</term><term>Spécificité du substrat</term><term>Thermodynamique</term><term>Transfert d'énergie</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Severe acute respiratory syndrome (SARS) coronavirus is a novel human coronavirus and is responsible for SARS infection. SARS coronavirus 3C-like proteinase (SARS 3CL(pro)) plays key roles in viral replication and transcription and is an attractive target for anti-SARS drug discovery. In this report, we quantitatively characterized the dimerization features of the full-length and N-terminal residues 1-7 deleted SARS 3CL(pro)s by using glutaraldehyde cross-linking SDS-PAGE, size-exclusion chromatography, and isothermal titration calorimeter techniques. Glutaraldehyde cross-linking SDS-PAGE and size-exclusion chromatography results show that, similar to the full-length SARS 3CL(pro), the N-terminal deleted SARS 3CL(pro) still remains a dimer/monomer mixture within a wide range of protein concentrations. Isothermal titration calorimeter determinations indicate that the equilibrium dissociation constant (K(d)) of the N-terminal deleted proteinase dimer (262 microm) is very similar to that of the full-length proteinase dimer (227 microm). Enzymatic activity assay using the fluorescence resonance energy transfer method reveals that N-terminal deletion results in almost complete loss of enzymatic activity for SARS 3CL(pro). Molecular dynamics and docking simulations demonstrate the N-terminal deleted proteinase dimer adopts a state different from that of the full-length proteinase dimer, which increases the angle between the two protomers and reduces the binding pocket that is not beneficial to the substrate binding. This conclusion is verified by the surface plasmon resonance biosensor determination, indicating that the model substrate cannot bind to the N-terminal deleted proteinase. These results suggest the N terminus is not indispensable for the proteinase dimerization but may fix the dimer at the active state and is therefore vital to enzymatic activity.</div></front></TEI><affiliations><list><country><li>République populaire de Chine</li></country></list><tree><noCountry><name sortKey="Chen, Jing" sort="Chen, Jing" uniqKey="Chen J" first="Jing" last="Chen">Jing Chen</name><name sortKey="Chen, Kaixian" sort="Chen, Kaixian" uniqKey="Chen K" first="Kaixian" last="Chen">Kaixian Chen</name><name sortKey="Chen, Lili" sort="Chen, Lili" uniqKey="Chen L" first="Lili" last="Chen">Lili Chen</name><name sortKey="Du, Li" sort="Du, Li" uniqKey="Du L" first="Li" last="Du">Li Du</name><name sortKey="Jiang, Hualiang" sort="Jiang, Hualiang" uniqKey="Jiang H" first="Hualiang" last="Jiang">Hualiang Jiang</name><name sortKey="Shen, Jianhua" sort="Shen, Jianhua" uniqKey="Shen J" first="Jianhua" last="Shen">Jianhua Shen</name><name sortKey="Shen, Xu" sort="Shen, Xu" uniqKey="Shen X" first="Xu" last="Shen">Xu Shen</name><name sortKey="Sun, Tao" sort="Sun, Tao" uniqKey="Sun T" first="Tao" last="Sun">Tao Sun</name><name sortKey="Tan, Jinzhi" sort="Tan, Jinzhi" uniqKey="Tan J" first="Jinzhi" last="Tan">Jinzhi Tan</name></noCountry><country name="République populaire de Chine"><noRegion><name sortKey="Chen, Shuai" sort="Chen, Shuai" uniqKey="Chen S" first="Shuai" last="Chen">Shuai Chen</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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