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 : 002A79 ( PubMed/Corpus ); précédent : 002A78; suivant : 002A80Severe 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 ; 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.
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
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pubmed:15507456Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">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.</title><author><name sortKey="Chen, Shuai" sort="Chen, Shuai" uniqKey="Chen S" first="Shuai" last="Chen">Shuai Chen</name><affiliation><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></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></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2005">2005</date><idno type="RBID">pubmed:15507456</idno><idno type="pmid">15507456</idno><idno type="doi">10.1074/jbc.M408211200</idno><idno type="wicri:Area/PubMed/Corpus">002A79</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Corpus" wicri:corpus="PubMed">002A79</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">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.</title><author><name sortKey="Chen, Shuai" sort="Chen, Shuai" uniqKey="Chen S" first="Shuai" last="Chen">Shuai Chen</name><affiliation><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></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="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="enzymology" xml:lang="en"><term>SARS Virus</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></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><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">15507456</PMID><DateCompleted><Year>2005</Year><Month>07</Month><Day>21</Day></DateCompleted><DateRevised><Year>2020</Year><Month>03</Month><Day>25</Day></DateRevised><Article PubModel="Print-Electronic"><Journal><ISSN IssnType="Print">0021-9258</ISSN><JournalIssue CitedMedium="Print"><Volume>280</Volume><Issue>1</Issue><PubDate><Year>2005</Year><Month>Jan</Month><Day>07</Day></PubDate></JournalIssue><Title>The Journal of biological chemistry</Title><ISOAbbreviation>J. Biol. Chem.</ISOAbbreviation></Journal><ArticleTitle>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.</ArticleTitle><Pagination><MedlinePgn>164-73</MedlinePgn></Pagination><Abstract><AbstractText>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.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Chen</LastName><ForeName>Shuai</ForeName><Initials>S</Initials><AffiliationInfo><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.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Chen</LastName><ForeName>Lili</ForeName><Initials>L</Initials></Author><Author ValidYN="Y"><LastName>Tan</LastName><ForeName>Jinzhi</ForeName><Initials>J</Initials></Author><Author ValidYN="Y"><LastName>Chen</LastName><ForeName>Jing</ForeName><Initials>J</Initials></Author><Author ValidYN="Y"><LastName>Du</LastName><ForeName>Li</ForeName><Initials>L</Initials></Author><Author ValidYN="Y"><LastName>Sun</LastName><ForeName>Tao</ForeName><Initials>T</Initials></Author><Author ValidYN="Y"><LastName>Shen</LastName><ForeName>Jianhua</ForeName><Initials>J</Initials></Author><Author ValidYN="Y"><LastName>Chen</LastName><ForeName>Kaixian</ForeName><Initials>K</Initials></Author><Author ValidYN="Y"><LastName>Jiang</LastName><ForeName>Hualiang</ForeName><Initials>H</Initials></Author><Author ValidYN="Y"><LastName>Shen</LastName><ForeName>Xu</ForeName><Initials>X</Initials></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013485">Research Support, Non-U.S. Gov't</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2004</Year><Month>10</Month><Day>26</Day></ArticleDate></Article><MedlineJournalInfo><Country>United States</Country><MedlineTA>J Biol Chem</MedlineTA><NlmUniqueID>2985121R</NlmUniqueID><ISSNLinking>0021-9258</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D014764">Viral Proteins</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 3.4.-</RegistryNumber><NameOfSubstance UI="D010450">Endopeptidases</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 3.4.22.-</RegistryNumber><NameOfSubstance UI="C099456">3C-like proteinase, Coronavirus</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 3.4.22.-</RegistryNumber><NameOfSubstance UI="D003546">Cysteine Endopeptidases</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D001665" MajorTopicYN="N">Binding Sites</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D002151" MajorTopicYN="N">Calorimetry</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D003546" MajorTopicYN="N">Cysteine Endopeptidases</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D019281" MajorTopicYN="N">Dimerization</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D010450" MajorTopicYN="Y">Endopeptidases</DescriptorName><QualifierName UI="Q000737" MajorTopicYN="N">chemistry</QualifierName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D004735" MajorTopicYN="N">Energy Transfer</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D011401" MajorTopicYN="N">Promoter Regions, Genetic</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D011487" MajorTopicYN="N">Protein Conformation</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D045473" MajorTopicYN="N">SARS Virus</DescriptorName><QualifierName UI="Q000201" MajorTopicYN="Y">enzymology</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D013379" MajorTopicYN="N">Substrate Specificity</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D013816" MajorTopicYN="N">Thermodynamics</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D014764" MajorTopicYN="Y">Viral Proteins</DescriptorName><QualifierName UI="Q000737" MajorTopicYN="N">chemistry</QualifierName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName></MeshHeading></MeshHeadingList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="pubmed"><Year>2004</Year><Month>10</Month><Day>28</Day><Hour>9</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>2005</Year><Month>7</Month><Day>22</Day><Hour>9</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>2004</Year><Month>10</Month><Day>28</Day><Hour>9</Hour><Minute>0</Minute></PubMedPubDate></History><PublicationStatus>ppublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">15507456</ArticleId><ArticleId IdType="pii">M408211200</ArticleId><ArticleId IdType="doi">10.1074/jbc.M408211200</ArticleId></ArticleIdList></PubmedData></pubmed></record>Pour manipuler ce document sous Unix (Dilib)
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