Kinetic, Mutational, and Structural Studies of the Venezuelan Equine Encephalitis Virus Nonstructural Protein 2 Cysteine Protease.
Identifieur interne : 001185 ( Main/Exploration ); précédent : 001184; suivant : 001186Kinetic, Mutational, and Structural Studies of the Venezuelan Equine Encephalitis Virus Nonstructural Protein 2 Cysteine Protease.
Auteurs : Xin Hu [États-Unis] ; Jaimee R. Compton [États-Unis] ; Dagmar H. Leary [États-Unis] ; Mark A. Olson [États-Unis] ; Michael S. Lee [États-Unis] ; Jonah Cheung [États-Unis] ; Wenjuan Ye [États-Unis] ; Mark Ferrer [États-Unis] ; Noel Southall [États-Unis] ; Ajit Jadhav [États-Unis] ; Elaine M. Morazzani [États-Unis] ; Pamela J. Glass [États-Unis] ; Juan Marugan [États-Unis] ; Patricia M. Legler [États-Unis]Source :
- Biochemistry [ 1520-4995 ] ; 2016.
Descripteurs français
- KwdFr :
- Adémétionine (métabolisme), Cinétique, Conformation des protéines, Cristallographie aux rayons X, Cysteine endopeptidases (), Cysteine endopeptidases (génétique), Cysteine endopeptidases (métabolisme), Domaine catalytique, Hydrolyse, Modèles moléculaires, Mutation (génétique), Papaïne (métabolisme), Protéines virales non structurales (), Protéines virales non structurales (génétique), Protéines virales non structurales (métabolisme), Similitude de séquences d'acides aminés, Sites de fixation, Séquence d'acides aminés, Virus de l'encéphalite équine du Venezuela (enzymologie).
- MESH :
- enzymologie : Virus de l'encéphalite équine du Venezuela.
- génétique : Cysteine endopeptidases, Mutation, Protéines virales non structurales.
- métabolisme : Adémétionine, Cysteine endopeptidases, Papaïne, Protéines virales non structurales.
- Cinétique, Conformation des protéines, Cristallographie aux rayons X, Cysteine endopeptidases, Domaine catalytique, Hydrolyse, Modèles moléculaires, Protéines virales non structurales, Similitude de séquences d'acides aminés, Sites de fixation, Séquence d'acides aminés.
English descriptors
- KwdEn :
- Amino Acid Sequence, Binding Sites, Catalytic Domain, Crystallography, X-Ray, Cysteine Endopeptidases (chemistry), Cysteine Endopeptidases (genetics), Cysteine Endopeptidases (metabolism), Encephalitis Virus, Venezuelan Equine (enzymology), Hydrolysis, Kinetics, Models, Molecular, Mutation (genetics), Papain (metabolism), Protein Conformation, S-Adenosylmethionine (metabolism), Sequence Homology, Amino Acid, Viral Nonstructural Proteins (chemistry), Viral Nonstructural Proteins (genetics), Viral Nonstructural Proteins (metabolism).
- MESH :
- chemical , chemistry : Cysteine Endopeptidases, Viral Nonstructural Proteins.
- chemical , genetics : Cysteine Endopeptidases, Viral Nonstructural Proteins.
- chemical , metabolism : Cysteine Endopeptidases, Papain, S-Adenosylmethionine, Viral Nonstructural Proteins.
- enzymology : Encephalitis Virus, Venezuelan Equine.
- genetics : Mutation.
- Amino Acid Sequence, Binding Sites, Catalytic Domain, Crystallography, X-Ray, Hydrolysis, Kinetics, Models, Molecular, Protein Conformation, Sequence Homology, Amino Acid.
Abstract
The Venezuelan equine encephalitis virus (VEEV) nonstructural protein 2 (nsP2) cysteine protease (EC 3.4.22.-) is essential for viral replication and is involved in the cytopathic effects (CPE) of the virus. The VEEV nsP2 protease is a member of MEROPS Clan CN and characteristically contains a papain-like protease linked to an S-adenosyl-l-methionine-dependent RNA methyltransferase (SAM MTase) domain. The protease contains an alternative active site motif, (475)NVCWAK(480), which differs from papain's (CGS(25)CWAFS), and the enzyme lacks a transition state-stabilizing residue homologous to Gln-19 in papain. To understand the roles of conserved residues in catalysis, we determined the structure of the free enzyme and the first structure of an inhibitor-bound alphaviral protease. The peptide-like E64d inhibitor was found to bind beneath a β-hairpin at the interface of the SAM MTase and protease domains. His-546 adopted a conformation that differed from that found in the free enzyme; one or both of the conformers may assist in leaving group departure of either the amine or Cys thiolate during the catalytic cycle. Interestingly, E64c (200 μM), the carboxylic acid form of the E64d ester, did not inhibit the nsP2 protease. To identify key residues involved in substrate binding, a number of mutants were analyzed. Mutation of the motif residue, N475A, led to a 24-fold reduction in kcat/Km, and the conformation of this residue did not change after inhibition. N475 forms a hydrogen bond with R662 in the SAM MTase domain, and the R662A and R662K mutations both led to 16-fold decreases in kcat/Km. N475 forms the base of the P1 binding site and likely orients the substrate for nucleophilic attack or plays a role in product release. An Asn homologous to N475 is similarly found in coronaviral papain-like proteases (PLpro) of the Severe Acute Respiratory Syndrome (SARS) virus and Middle East Respiratory Syndrome (MERS) virus. Mutation of another motif residue, K480A, led to a 9-fold decrease in kcat and kcat/Km. K480 likely enhances the nucleophilicity of the Cys. Consistent with our substrate-bound models, the SAM MTase domain K706A mutation increased Km 4.5-fold to 500 μM. Within the β-hairpin, the N545A mutation slightly but not significantly increased kcat and Km. The structures and identified active site residues may facilitate the discovery of protease inhibitors with antiviral activity.
DOI: 10.1021/acs.biochem.5b00992
PubMed: 27030368
Affiliations:
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Le document en format XML
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<sourceDesc><biblStruct><analytic><title xml:lang="en">Kinetic, Mutational, and Structural Studies of the Venezuelan Equine Encephalitis Virus Nonstructural Protein 2 Cysteine Protease.</title>
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<author><name sortKey="Olson, Mark A" sort="Olson, Mark A" uniqKey="Olson M" first="Mark A" last="Olson">Mark A. Olson</name>
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<country xml:lang="fr">États-Unis</country>
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<author><name sortKey="Lee, Michael S" sort="Lee, Michael S" uniqKey="Lee M" first="Michael S" last="Lee">Michael S. Lee</name>
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<author><name sortKey="Cheung, Jonah" sort="Cheung, Jonah" uniqKey="Cheung J" first="Jonah" last="Cheung">Jonah Cheung</name>
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<country xml:lang="fr">États-Unis</country>
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<wicri:noRegion>New York 10027</wicri:noRegion>
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<author><name sortKey="Ye, Wenjuan" sort="Ye, Wenjuan" uniqKey="Ye W" first="Wenjuan" last="Ye">Wenjuan Ye</name>
<affiliation wicri:level="1"><nlm:affiliation>NIH Chemical Genomics Center, National Center for Advancing Translational Sciences , Rockville, Maryland 20850, United States.</nlm:affiliation>
<country xml:lang="fr">États-Unis</country>
<wicri:regionArea>NIH Chemical Genomics Center, National Center for Advancing Translational Sciences , Rockville, Maryland 20850</wicri:regionArea>
<wicri:noRegion>Maryland 20850</wicri:noRegion>
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<author><name sortKey="Ferrer, Mark" sort="Ferrer, Mark" uniqKey="Ferrer M" first="Mark" last="Ferrer">Mark Ferrer</name>
<affiliation wicri:level="1"><nlm:affiliation>NIH Chemical Genomics Center, National Center for Advancing Translational Sciences , Rockville, Maryland 20850, United States.</nlm:affiliation>
<country xml:lang="fr">États-Unis</country>
<wicri:regionArea>NIH Chemical Genomics Center, National Center for Advancing Translational Sciences , Rockville, Maryland 20850</wicri:regionArea>
<wicri:noRegion>Maryland 20850</wicri:noRegion>
</affiliation>
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<author><name sortKey="Southall, Noel" sort="Southall, Noel" uniqKey="Southall N" first="Noel" last="Southall">Noel Southall</name>
<affiliation wicri:level="1"><nlm:affiliation>NIH Chemical Genomics Center, National Center for Advancing Translational Sciences , Rockville, Maryland 20850, United States.</nlm:affiliation>
<country xml:lang="fr">États-Unis</country>
<wicri:regionArea>NIH Chemical Genomics Center, National Center for Advancing Translational Sciences , Rockville, Maryland 20850</wicri:regionArea>
<wicri:noRegion>Maryland 20850</wicri:noRegion>
</affiliation>
</author>
<author><name sortKey="Jadhav, Ajit" sort="Jadhav, Ajit" uniqKey="Jadhav A" first="Ajit" last="Jadhav">Ajit Jadhav</name>
<affiliation wicri:level="1"><nlm:affiliation>NIH Chemical Genomics Center, National Center for Advancing Translational Sciences , Rockville, Maryland 20850, United States.</nlm:affiliation>
<country xml:lang="fr">États-Unis</country>
<wicri:regionArea>NIH Chemical Genomics Center, National Center for Advancing Translational Sciences , Rockville, Maryland 20850</wicri:regionArea>
<wicri:noRegion>Maryland 20850</wicri:noRegion>
</affiliation>
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<author><name sortKey="Morazzani, Elaine M" sort="Morazzani, Elaine M" uniqKey="Morazzani E" first="Elaine M" last="Morazzani">Elaine M. Morazzani</name>
<affiliation wicri:level="1"><nlm:affiliation>United States Army Medical Research Institute of Infectious Diseases , Frederick, Maryland 21702, United States.</nlm:affiliation>
<country xml:lang="fr">États-Unis</country>
<wicri:regionArea>United States Army Medical Research Institute of Infectious Diseases , Frederick, Maryland 21702</wicri:regionArea>
<wicri:noRegion>Maryland 21702</wicri:noRegion>
</affiliation>
</author>
<author><name sortKey="Glass, Pamela J" sort="Glass, Pamela J" uniqKey="Glass P" first="Pamela J" last="Glass">Pamela J. Glass</name>
<affiliation wicri:level="1"><nlm:affiliation>United States Army Medical Research Institute of Infectious Diseases , Frederick, Maryland 21702, United States.</nlm:affiliation>
<country xml:lang="fr">États-Unis</country>
<wicri:regionArea>United States Army Medical Research Institute of Infectious Diseases , Frederick, Maryland 21702</wicri:regionArea>
<wicri:noRegion>Maryland 21702</wicri:noRegion>
</affiliation>
</author>
<author><name sortKey="Marugan, Juan" sort="Marugan, Juan" uniqKey="Marugan J" first="Juan" last="Marugan">Juan Marugan</name>
<affiliation wicri:level="1"><nlm:affiliation>NIH Chemical Genomics Center, National Center for Advancing Translational Sciences , Rockville, Maryland 20850, United States.</nlm:affiliation>
<country xml:lang="fr">États-Unis</country>
<wicri:regionArea>NIH Chemical Genomics Center, National Center for Advancing Translational Sciences , Rockville, Maryland 20850</wicri:regionArea>
<wicri:noRegion>Maryland 20850</wicri:noRegion>
</affiliation>
</author>
<author><name sortKey="Legler, Patricia M" sort="Legler, Patricia M" uniqKey="Legler P" first="Patricia M" last="Legler">Patricia M. Legler</name>
<affiliation wicri:level="1"><nlm:affiliation>Center for Bio/molecular Science and Engineering, U.S. Naval Research Laboratory , Washington, D.C. 20375, United States.</nlm:affiliation>
<country xml:lang="fr">États-Unis</country>
<wicri:regionArea>Center for Bio/molecular Science and Engineering, U.S. Naval Research Laboratory , Washington, D.C. 20375</wicri:regionArea>
<wicri:noRegion>D.C. 20375</wicri:noRegion>
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<series><title level="j">Biochemistry</title>
<idno type="eISSN">1520-4995</idno>
<imprint><date when="2016" type="published">2016</date>
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<profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Amino Acid Sequence</term>
<term>Binding Sites</term>
<term>Catalytic Domain</term>
<term>Crystallography, X-Ray</term>
<term>Cysteine Endopeptidases (chemistry)</term>
<term>Cysteine Endopeptidases (genetics)</term>
<term>Cysteine Endopeptidases (metabolism)</term>
<term>Encephalitis Virus, Venezuelan Equine (enzymology)</term>
<term>Hydrolysis</term>
<term>Kinetics</term>
<term>Models, Molecular</term>
<term>Mutation (genetics)</term>
<term>Papain (metabolism)</term>
<term>Protein Conformation</term>
<term>S-Adenosylmethionine (metabolism)</term>
<term>Sequence Homology, Amino Acid</term>
<term>Viral Nonstructural Proteins (chemistry)</term>
<term>Viral Nonstructural Proteins (genetics)</term>
<term>Viral Nonstructural Proteins (metabolism)</term>
</keywords>
<keywords scheme="KwdFr" xml:lang="fr"><term>Adémétionine (métabolisme)</term>
<term>Cinétique</term>
<term>Conformation des protéines</term>
<term>Cristallographie aux rayons X</term>
<term>Cysteine endopeptidases ()</term>
<term>Cysteine endopeptidases (génétique)</term>
<term>Cysteine endopeptidases (métabolisme)</term>
<term>Domaine catalytique</term>
<term>Hydrolyse</term>
<term>Modèles moléculaires</term>
<term>Mutation (génétique)</term>
<term>Papaïne (métabolisme)</term>
<term>Protéines virales non structurales ()</term>
<term>Protéines virales non structurales (génétique)</term>
<term>Protéines virales non structurales (métabolisme)</term>
<term>Similitude de séquences d'acides aminés</term>
<term>Sites de fixation</term>
<term>Séquence d'acides aminés</term>
<term>Virus de l'encéphalite équine du Venezuela (enzymologie)</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Cysteine Endopeptidases</term>
<term>Viral Nonstructural Proteins</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Cysteine Endopeptidases</term>
<term>Viral Nonstructural Proteins</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Cysteine Endopeptidases</term>
<term>Papain</term>
<term>S-Adenosylmethionine</term>
<term>Viral Nonstructural Proteins</term>
</keywords>
<keywords scheme="MESH" qualifier="enzymologie" xml:lang="fr"><term>Virus de l'encéphalite équine du Venezuela</term>
</keywords>
<keywords scheme="MESH" qualifier="enzymology" xml:lang="en"><term>Encephalitis Virus, Venezuelan Equine</term>
</keywords>
<keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Mutation</term>
</keywords>
<keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Cysteine endopeptidases</term>
<term>Mutation</term>
<term>Protéines virales non structurales</term>
</keywords>
<keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Adémétionine</term>
<term>Cysteine endopeptidases</term>
<term>Papaïne</term>
<term>Protéines virales non structurales</term>
</keywords>
<keywords scheme="MESH" xml:lang="en"><term>Amino Acid Sequence</term>
<term>Binding Sites</term>
<term>Catalytic Domain</term>
<term>Crystallography, X-Ray</term>
<term>Hydrolysis</term>
<term>Kinetics</term>
<term>Models, Molecular</term>
<term>Protein Conformation</term>
<term>Sequence Homology, Amino Acid</term>
</keywords>
<keywords scheme="MESH" xml:lang="fr"><term>Cinétique</term>
<term>Conformation des protéines</term>
<term>Cristallographie aux rayons X</term>
<term>Cysteine endopeptidases</term>
<term>Domaine catalytique</term>
<term>Hydrolyse</term>
<term>Modèles moléculaires</term>
<term>Protéines virales non structurales</term>
<term>Similitude de séquences d'acides aminés</term>
<term>Sites de fixation</term>
<term>Séquence d'acides aminés</term>
</keywords>
</textClass>
</profileDesc>
</teiHeader>
<front><div type="abstract" xml:lang="en">The Venezuelan equine encephalitis virus (VEEV) nonstructural protein 2 (nsP2) cysteine protease (EC 3.4.22.-) is essential for viral replication and is involved in the cytopathic effects (CPE) of the virus. The VEEV nsP2 protease is a member of MEROPS Clan CN and characteristically contains a papain-like protease linked to an S-adenosyl-l-methionine-dependent RNA methyltransferase (SAM MTase) domain. The protease contains an alternative active site motif, (475)NVCWAK(480), which differs from papain's (CGS(25)CWAFS), and the enzyme lacks a transition state-stabilizing residue homologous to Gln-19 in papain. To understand the roles of conserved residues in catalysis, we determined the structure of the free enzyme and the first structure of an inhibitor-bound alphaviral protease. The peptide-like E64d inhibitor was found to bind beneath a β-hairpin at the interface of the SAM MTase and protease domains. His-546 adopted a conformation that differed from that found in the free enzyme; one or both of the conformers may assist in leaving group departure of either the amine or Cys thiolate during the catalytic cycle. Interestingly, E64c (200 μM), the carboxylic acid form of the E64d ester, did not inhibit the nsP2 protease. To identify key residues involved in substrate binding, a number of mutants were analyzed. Mutation of the motif residue, N475A, led to a 24-fold reduction in kcat/Km, and the conformation of this residue did not change after inhibition. N475 forms a hydrogen bond with R662 in the SAM MTase domain, and the R662A and R662K mutations both led to 16-fold decreases in kcat/Km. N475 forms the base of the P1 binding site and likely orients the substrate for nucleophilic attack or plays a role in product release. An Asn homologous to N475 is similarly found in coronaviral papain-like proteases (PLpro) of the Severe Acute Respiratory Syndrome (SARS) virus and Middle East Respiratory Syndrome (MERS) virus. Mutation of another motif residue, K480A, led to a 9-fold decrease in kcat and kcat/Km. K480 likely enhances the nucleophilicity of the Cys. Consistent with our substrate-bound models, the SAM MTase domain K706A mutation increased Km 4.5-fold to 500 μM. Within the β-hairpin, the N545A mutation slightly but not significantly increased kcat and Km. The structures and identified active site residues may facilitate the discovery of protease inhibitors with antiviral activity.</div>
</front>
</TEI>
<affiliations><list><country><li>États-Unis</li>
</country>
</list>
<tree><country name="États-Unis"><noRegion><name sortKey="Hu, Xin" sort="Hu, Xin" uniqKey="Hu X" first="Xin" last="Hu">Xin Hu</name>
</noRegion>
<name sortKey="Cheung, Jonah" sort="Cheung, Jonah" uniqKey="Cheung J" first="Jonah" last="Cheung">Jonah Cheung</name>
<name sortKey="Compton, Jaimee R" sort="Compton, Jaimee R" uniqKey="Compton J" first="Jaimee R" last="Compton">Jaimee R. Compton</name>
<name sortKey="Ferrer, Mark" sort="Ferrer, Mark" uniqKey="Ferrer M" first="Mark" last="Ferrer">Mark Ferrer</name>
<name sortKey="Glass, Pamela J" sort="Glass, Pamela J" uniqKey="Glass P" first="Pamela J" last="Glass">Pamela J. Glass</name>
<name sortKey="Jadhav, Ajit" sort="Jadhav, Ajit" uniqKey="Jadhav A" first="Ajit" last="Jadhav">Ajit Jadhav</name>
<name sortKey="Leary, Dagmar H" sort="Leary, Dagmar H" uniqKey="Leary D" first="Dagmar H" last="Leary">Dagmar H. Leary</name>
<name sortKey="Lee, Michael S" sort="Lee, Michael S" uniqKey="Lee M" first="Michael S" last="Lee">Michael S. Lee</name>
<name sortKey="Legler, Patricia M" sort="Legler, Patricia M" uniqKey="Legler P" first="Patricia M" last="Legler">Patricia M. Legler</name>
<name sortKey="Marugan, Juan" sort="Marugan, Juan" uniqKey="Marugan J" first="Juan" last="Marugan">Juan Marugan</name>
<name sortKey="Morazzani, Elaine M" sort="Morazzani, Elaine M" uniqKey="Morazzani E" first="Elaine M" last="Morazzani">Elaine M. Morazzani</name>
<name sortKey="Olson, Mark A" sort="Olson, Mark A" uniqKey="Olson M" first="Mark A" last="Olson">Mark A. Olson</name>
<name sortKey="Southall, Noel" sort="Southall, Noel" uniqKey="Southall N" first="Noel" last="Southall">Noel Southall</name>
<name sortKey="Ye, Wenjuan" sort="Ye, Wenjuan" uniqKey="Ye W" first="Wenjuan" last="Ye">Wenjuan Ye</name>
</country>
</tree>
</affiliations>
</record>
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