Mutation of Asn28 disrupts the dimerization and enzymatic activity of SARS 3CL(pro) .
Identifieur interne : 002496 ( Main/Exploration ); précédent : 002495; suivant : 002497Mutation of Asn28 disrupts the dimerization and enzymatic activity of SARS 3CL(pro) .
Auteurs : Jennifer Barrila [États-Unis] ; Sandra B. Gabelli ; Usman Bacha ; L Mario Amzel ; Ernesto FreireSource :
- Biochemistry [ 1520-4995 ] ; 2010.
Descripteurs français
- KwdFr :
- Activation enzymatique (génétique), Asparagine (), Asparagine (génétique), Catalyse, Clonage moléculaire, Cristallographie aux rayons X, Cysteine endopeptidases (), Cysteine endopeptidases (génétique), Cysteine endopeptidases (métabolisme), Données de séquences moléculaires, Modèles moléculaires, Motifs et domaines d'intéraction protéique (génétique), Multimérisation de protéines (génétique), Mutation (physiologie), Protéines virales (), Protéines virales (génétique), Protéines virales (métabolisme), Similitude de séquences d'acides aminés, Substitution d'acide aminé (génétique), Substitution d'acide aminé (physiologie), Séquence d'acides aminés, Virus du SRAS (génétique), Virus du SRAS (métabolisme).
- MESH :
- génétique : Activation enzymatique, Asparagine, Cysteine endopeptidases, Motifs et domaines d'intéraction protéique, Multimérisation de protéines, Protéines virales, Substitution d'acide aminé, Virus du SRAS.
- métabolisme : Cysteine endopeptidases, Protéines virales, Virus du SRAS.
- physiologie : Mutation, Substitution d'acide aminé.
- Asparagine, Catalyse, Clonage moléculaire, Cristallographie aux rayons X, Cysteine endopeptidases, Données de séquences moléculaires, Modèles moléculaires, Protéines virales, Similitude de séquences d'acides aminés, Séquence d'acides aminés.
English descriptors
- KwdEn :
- Amino Acid Sequence, Amino Acid Substitution (genetics), Amino Acid Substitution (physiology), Asparagine (chemistry), Asparagine (genetics), Catalysis, Cloning, Molecular, Crystallography, X-Ray, Cysteine Endopeptidases (chemistry), Cysteine Endopeptidases (genetics), Cysteine Endopeptidases (metabolism), Enzyme Activation (genetics), Models, Molecular, Molecular Sequence Data, Mutation (physiology), Protein Interaction Domains and Motifs (genetics), Protein Multimerization (genetics), SARS Virus (genetics), SARS Virus (metabolism), Sequence Homology, Amino Acid, Viral Proteins (chemistry), Viral Proteins (genetics), Viral Proteins (metabolism).
- MESH :
- chemical , chemistry : Asparagine, Cysteine Endopeptidases, Viral Proteins.
- genetics : Amino Acid Substitution, Asparagine, Cysteine Endopeptidases, Enzyme Activation, Protein Interaction Domains and Motifs, Protein Multimerization, SARS Virus, Viral Proteins.
- chemical , metabolism : Cysteine Endopeptidases, SARS Virus, Viral Proteins.
- physiology : Amino Acid Substitution, Mutation.
- Amino Acid Sequence, Catalysis, Cloning, Molecular, Crystallography, X-Ray, Models, Molecular, Molecular Sequence Data, Sequence Homology, Amino Acid.
Abstract
Coronaviruses are responsible for a significant proportion of annual respiratory and enteric infections in humans and other mammals. The most prominent of these viruses is the severe acute respiratory syndrome coronavirus (SARS-CoV) which causes acute respiratory and gastrointestinal infection in humans. The coronavirus main protease, 3CL(pro), is a key target for broad-spectrum antiviral development because of its critical role in viral maturation and high degree of structural conservation among coronaviruses. Dimerization is an indispensable requirement for the function of SARS 3CL(pro) and is regulated through mechanisms involving both direct and long-range interactions in the enzyme. While many of the binding interactions at the dimerization interface have been extensively studied, those that are important for long-range control are not well-understood. Characterization of these dimerization mechanisms is important for the structure-based design of new treatments targeting coronavirus-based infections. Here we report that Asn28, a residue 11 A from the closest residue in the opposing monomer, is essential for the enzymatic activity and dimerization of SARS 3CL(pro). Mutation of this residue to alanine almost completely inactivates the enzyme and results in a 19.2-fold decrease in the dimerization K(d). The crystallographic structure of the N28A mutant determined at 2.35 A resolution reveals the critical role of Asn28 in maintaining the structural integrity of the active site and in orienting key residues involved in binding at the dimer interface and substrate catalysis. These findings provide deeper insight into complex mechanisms regulating the activity and dimerization of SARS 3CL(pro).
DOI: 10.1021/bi1002585
PubMed: 20420403
Affiliations:
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Le document en format XML
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Gabelli</name></author><author><name sortKey="Bacha, Usman" sort="Bacha, Usman" uniqKey="Bacha U" first="Usman" last="Bacha">Usman Bacha</name></author><author><name sortKey="Amzel, L Mario" sort="Amzel, L Mario" uniqKey="Amzel L" first="L Mario" last="Amzel">L Mario Amzel</name></author><author><name sortKey="Freire, Ernesto" sort="Freire, Ernesto" uniqKey="Freire E" first="Ernesto" last="Freire">Ernesto Freire</name></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2010">2010</date><idno type="RBID">pubmed:20420403</idno><idno type="pmid">20420403</idno><idno type="doi">10.1021/bi1002585</idno><idno type="wicri:Area/PubMed/Corpus">001706</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Corpus" wicri:corpus="PubMed">001706</idno><idno type="wicri:Area/PubMed/Curation">001706</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Curation">001706</idno><idno type="wicri:Area/PubMed/Checkpoint">001629</idno><idno type="wicri:explorRef" wicri:stream="Checkpoint" wicri:step="PubMed">001629</idno><idno type="wicri:Area/Ncbi/Merge">002128</idno><idno type="wicri:Area/Ncbi/Curation">002128</idno><idno type="wicri:Area/Ncbi/Checkpoint">002128</idno><idno type="wicri:Area/Main/Merge">002529</idno><idno type="wicri:Area/Main/Curation">002496</idno><idno type="wicri:Area/Main/Exploration">002496</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">Mutation of Asn28 disrupts the dimerization and enzymatic activity of SARS 3CL(pro) .</title><author><name sortKey="Barrila, Jennifer" sort="Barrila, Jennifer" uniqKey="Barrila J" first="Jennifer" last="Barrila">Jennifer Barrila</name><affiliation wicri:level="1"><nlm:affiliation>Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218, USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218</wicri:regionArea><wicri:noRegion>Maryland 21218</wicri:noRegion></affiliation></author><author><name sortKey="Gabelli, Sandra B" sort="Gabelli, Sandra B" uniqKey="Gabelli S" first="Sandra B" last="Gabelli">Sandra B. Gabelli</name></author><author><name sortKey="Bacha, Usman" sort="Bacha, Usman" uniqKey="Bacha U" first="Usman" last="Bacha">Usman Bacha</name></author><author><name sortKey="Amzel, L Mario" sort="Amzel, L Mario" uniqKey="Amzel L" first="L Mario" last="Amzel">L Mario Amzel</name></author><author><name sortKey="Freire, Ernesto" sort="Freire, Ernesto" uniqKey="Freire E" first="Ernesto" last="Freire">Ernesto Freire</name></author></analytic><series><title level="j">Biochemistry</title><idno type="eISSN">1520-4995</idno><imprint><date when="2010" type="published">2010</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Amino Acid Sequence</term><term>Amino Acid Substitution (genetics)</term><term>Amino Acid Substitution (physiology)</term><term>Asparagine (chemistry)</term><term>Asparagine (genetics)</term><term>Catalysis</term><term>Cloning, Molecular</term><term>Crystallography, X-Ray</term><term>Cysteine Endopeptidases (chemistry)</term><term>Cysteine Endopeptidases (genetics)</term><term>Cysteine Endopeptidases (metabolism)</term><term>Enzyme Activation (genetics)</term><term>Models, Molecular</term><term>Molecular Sequence Data</term><term>Mutation (physiology)</term><term>Protein Interaction Domains and Motifs (genetics)</term><term>Protein Multimerization (genetics)</term><term>SARS Virus (genetics)</term><term>SARS Virus (metabolism)</term><term>Sequence Homology, Amino Acid</term><term>Viral Proteins (chemistry)</term><term>Viral Proteins (genetics)</term><term>Viral Proteins (metabolism)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Activation enzymatique (génétique)</term><term>Asparagine ()</term><term>Asparagine (génétique)</term><term>Catalyse</term><term>Clonage moléculaire</term><term>Cristallographie aux rayons X</term><term>Cysteine endopeptidases ()</term><term>Cysteine endopeptidases (génétique)</term><term>Cysteine endopeptidases (métabolisme)</term><term>Données de séquences moléculaires</term><term>Modèles moléculaires</term><term>Motifs et domaines d'intéraction protéique (génétique)</term><term>Multimérisation de protéines (génétique)</term><term>Mutation (physiologie)</term><term>Protéines virales ()</term><term>Protéines virales (génétique)</term><term>Protéines virales (métabolisme)</term><term>Similitude de séquences d'acides aminés</term><term>Substitution d'acide aminé (génétique)</term><term>Substitution d'acide aminé (physiologie)</term><term>Séquence d'acides aminés</term><term>Virus du SRAS (génétique)</term><term>Virus du SRAS (métabolisme)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Asparagine</term><term>Cysteine Endopeptidases</term><term>Viral Proteins</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Amino Acid Substitution</term><term>Asparagine</term><term>Cysteine Endopeptidases</term><term>Enzyme Activation</term><term>Protein Interaction Domains and Motifs</term><term>Protein Multimerization</term><term>SARS Virus</term><term>Viral Proteins</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Activation enzymatique</term><term>Asparagine</term><term>Cysteine endopeptidases</term><term>Motifs et domaines d'intéraction protéique</term><term>Multimérisation de protéines</term><term>Protéines virales</term><term>Substitution d'acide aminé</term><term>Virus du SRAS</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Cysteine Endopeptidases</term><term>SARS Virus</term><term>Viral Proteins</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Cysteine endopeptidases</term><term>Protéines virales</term><term>Virus du SRAS</term></keywords><keywords scheme="MESH" qualifier="physiologie" xml:lang="fr"><term>Mutation</term><term>Substitution d'acide aminé</term></keywords><keywords scheme="MESH" qualifier="physiology" xml:lang="en"><term>Amino Acid Substitution</term><term>Mutation</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Amino Acid Sequence</term><term>Catalysis</term><term>Cloning, Molecular</term><term>Crystallography, X-Ray</term><term>Models, Molecular</term><term>Molecular Sequence Data</term><term>Sequence Homology, Amino Acid</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Asparagine</term><term>Catalyse</term><term>Clonage moléculaire</term><term>Cristallographie aux rayons X</term><term>Cysteine endopeptidases</term><term>Données de séquences moléculaires</term><term>Modèles moléculaires</term><term>Protéines virales</term><term>Similitude de séquences d'acides aminés</term><term>Séquence d'acides aminés</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Coronaviruses are responsible for a significant proportion of annual respiratory and enteric infections in humans and other mammals. The most prominent of these viruses is the severe acute respiratory syndrome coronavirus (SARS-CoV) which causes acute respiratory and gastrointestinal infection in humans. The coronavirus main protease, 3CL(pro), is a key target for broad-spectrum antiviral development because of its critical role in viral maturation and high degree of structural conservation among coronaviruses. Dimerization is an indispensable requirement for the function of SARS 3CL(pro) and is regulated through mechanisms involving both direct and long-range interactions in the enzyme. While many of the binding interactions at the dimerization interface have been extensively studied, those that are important for long-range control are not well-understood. Characterization of these dimerization mechanisms is important for the structure-based design of new treatments targeting coronavirus-based infections. Here we report that Asn28, a residue 11 A from the closest residue in the opposing monomer, is essential for the enzymatic activity and dimerization of SARS 3CL(pro). Mutation of this residue to alanine almost completely inactivates the enzyme and results in a 19.2-fold decrease in the dimerization K(d). The crystallographic structure of the N28A mutant determined at 2.35 A resolution reveals the critical role of Asn28 in maintaining the structural integrity of the active site and in orienting key residues involved in binding at the dimer interface and substrate catalysis. These findings provide deeper insight into complex mechanisms regulating the activity and dimerization of SARS 3CL(pro).</div></front></TEI><affiliations><list><country><li>États-Unis</li></country></list><tree><noCountry><name sortKey="Amzel, L Mario" sort="Amzel, L Mario" uniqKey="Amzel L" first="L Mario" last="Amzel">L Mario Amzel</name><name sortKey="Bacha, Usman" sort="Bacha, Usman" uniqKey="Bacha U" first="Usman" last="Bacha">Usman Bacha</name><name sortKey="Freire, Ernesto" sort="Freire, Ernesto" uniqKey="Freire E" first="Ernesto" last="Freire">Ernesto Freire</name><name sortKey="Gabelli, Sandra B" sort="Gabelli, Sandra B" uniqKey="Gabelli S" first="Sandra B" last="Gabelli">Sandra B. Gabelli</name></noCountry><country name="États-Unis"><noRegion><name sortKey="Barrila, Jennifer" sort="Barrila, Jennifer" uniqKey="Barrila J" first="Jennifer" last="Barrila">Jennifer Barrila</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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