Mutation of Asn-475 in the Venezuelan Equine Encephalitis Virus nsP2 Cysteine Protease Leads to a Self-Inhibited State.
Identifieur interne : 000C68 ( Main/Exploration ); précédent : 000C67; suivant : 000C69Mutation of Asn-475 in the Venezuelan Equine Encephalitis Virus nsP2 Cysteine Protease Leads to a Self-Inhibited State.
Auteurs : Jaimee R. Compton [États-Unis] ; Matthew J. Mickey [États-Unis] ; Xin Hu [États-Unis] ; Juan J. Marugan [États-Unis] ; Patricia M. Legler [États-Unis]Source :
- Biochemistry [ 1520-4995 ] ; 2017.
Descripteurs français
- KwdFr :
- Conformation des protéines, Cristallographie aux rayons X, Cysteine endopeptidases (), Cysteine endopeptidases (génétique), Cysteine endopeptidases (métabolisme), Domaine catalytique, Fixation compétitive, Humains, Hydrolyse, Modèles moléculaires, Mutagenèse dirigée, Mutation, Protéines virales (), Protéines virales (génétique), Protéines virales (métabolisme), Rétrocontrôle physiologique, Similitude de séquences, 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, Protéines virales.
- métabolisme : Cysteine endopeptidases, Protéines virales.
- Conformation des protéines, Cristallographie aux rayons X, Cysteine endopeptidases, Domaine catalytique, Fixation compétitive, Humains, Hydrolyse, Modèles moléculaires, Mutagenèse dirigée, Mutation, Protéines virales, Rétrocontrôle physiologique, Similitude de séquences, Sites de fixation, Séquence d'acides aminés.
English descriptors
- KwdEn :
- Amino Acid Sequence, Binding Sites, Binding, Competitive, Catalytic Domain, Crystallography, X-Ray, Cysteine Endopeptidases (chemistry), Cysteine Endopeptidases (genetics), Cysteine Endopeptidases (metabolism), Encephalitis Virus, Venezuelan Equine (enzymology), Feedback, Physiological, Humans, Hydrolysis, Models, Molecular, Mutagenesis, Site-Directed, Mutation, Protein Conformation, Sequence Homology, Viral Proteins (chemistry), Viral Proteins (genetics), Viral Proteins (metabolism).
- MESH :
- chemical , chemistry : Cysteine Endopeptidases, Viral Proteins.
- chemical , genetics : Cysteine Endopeptidases, Viral Proteins.
- chemical , metabolism : Cysteine Endopeptidases, Viral Proteins.
- enzymology : Encephalitis Virus, Venezuelan Equine.
- Amino Acid Sequence, Binding Sites, Binding, Competitive, Catalytic Domain, Crystallography, X-Ray, Feedback, Physiological, Humans, Hydrolysis, Models, Molecular, Mutagenesis, Site-Directed, Mutation, Protein Conformation, Sequence Homology.
Abstract
The alphaviral nsP2 cysteine protease of the Venezuelan equine encephalitis virus (VEEV) is a validated antiviral drug target. Clan CN proteases contain a cysteine protease domain that is intimately packed with an S-adenosyl-l-methionine-dependent RNA methyltransferase (SAM MTase) domain. Within a cleft formed at the interface of these two domains, the peptide substrate is thought to bind. The nucleophilic cysteine can be found within a conserved motif, 475NVCWAK480, which differs from that of papain (22CGSCWAFS29). Mutation of the motif residue, N475, to alanine unexpectedly produced a self-inhibited state in which the N-terminal residues flipped into the substrate-binding cleft. Notably, the N-terminal segment was not hydrolyzed-consistent with a catalytically incompetent state. The N475A mutation resulted in a 70-fold decrease in kcat/Km. A side chain-substrate interaction was predicted by the structure; the S701A mutation led to a 17-fold increase in Km. An Asn at the n-2 position relative to the Cys was also found in the coronaviral papain-like proteases/deubiquitinases (PLpro) of the SARS and MERS viruses, and in several papain-like human ubiquitin specific proteases (USP). The large conformational change in the N475A variant suggests that Asn-475 plays an important role in stabilizing the N-terminal residues and in orienting the carbonyl during nucleophilic attack but does not directly hydrogen bond the oxyanion. The state trapped in crystallo is an unusual result of site-directed mutagenesis but reveals the role of this highly conserved Asn and identifies key substrate-binding contacts that may be exploited by peptide-like inhibitors.
DOI: 10.1021/acs.biochem.7b00746
PubMed: 29064679
Affiliations:
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Compton</name><affiliation wicri:level="1"><nlm:affiliation>U.S. Naval Research Laboratory , 4555 Overlook Avenue, Washington, D.C. 20375, United States.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>U.S. Naval Research Laboratory , 4555 Overlook Avenue, Washington, D.C. 20375</wicri:regionArea><wicri:noRegion>D.C. 20375</wicri:noRegion></affiliation></author><author><name sortKey="Mickey, Matthew J" sort="Mickey, Matthew J" uniqKey="Mickey M" first="Matthew J" last="Mickey">Matthew J. 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Legler</name><affiliation wicri:level="1"><nlm:affiliation>U.S. Naval Research Laboratory , 4555 Overlook Avenue, Washington, D.C. 20375, United States.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>U.S. Naval Research Laboratory , 4555 Overlook Avenue, Washington, D.C. 20375</wicri:regionArea><wicri:noRegion>D.C. 20375</wicri:noRegion></affiliation></author></analytic><series><title level="j">Biochemistry</title><idno type="eISSN">1520-4995</idno><imprint><date when="2017" type="published">2017</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Amino Acid Sequence</term><term>Binding Sites</term><term>Binding, Competitive</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>Feedback, Physiological</term><term>Humans</term><term>Hydrolysis</term><term>Models, Molecular</term><term>Mutagenesis, Site-Directed</term><term>Mutation</term><term>Protein Conformation</term><term>Sequence Homology</term><term>Viral Proteins (chemistry)</term><term>Viral Proteins (genetics)</term><term>Viral Proteins (metabolism)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><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>Fixation compétitive</term><term>Humains</term><term>Hydrolyse</term><term>Modèles moléculaires</term><term>Mutagenèse dirigée</term><term>Mutation</term><term>Protéines virales ()</term><term>Protéines virales (génétique)</term><term>Protéines virales (métabolisme)</term><term>Rétrocontrôle physiologique</term><term>Similitude de séquences</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 Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Cysteine Endopeptidases</term><term>Viral Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Cysteine Endopeptidases</term><term>Viral 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="génétique" xml:lang="fr"><term>Cysteine endopeptidases</term><term>Protéines virales</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Cysteine endopeptidases</term><term>Protéines virales</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Amino Acid Sequence</term><term>Binding Sites</term><term>Binding, Competitive</term><term>Catalytic Domain</term><term>Crystallography, X-Ray</term><term>Feedback, Physiological</term><term>Humans</term><term>Hydrolysis</term><term>Models, Molecular</term><term>Mutagenesis, Site-Directed</term><term>Mutation</term><term>Protein Conformation</term><term>Sequence Homology</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Conformation des protéines</term><term>Cristallographie aux rayons X</term><term>Cysteine endopeptidases</term><term>Domaine catalytique</term><term>Fixation compétitive</term><term>Humains</term><term>Hydrolyse</term><term>Modèles moléculaires</term><term>Mutagenèse dirigée</term><term>Mutation</term><term>Protéines virales</term><term>Rétrocontrôle physiologique</term><term>Similitude de séquences</term><term>Sites de fixation</term><term>Séquence d'acides aminés</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The alphaviral nsP2 cysteine protease of the Venezuelan equine encephalitis virus (VEEV) is a validated antiviral drug target. Clan CN proteases contain a cysteine protease domain that is intimately packed with an S-adenosyl-l-methionine-dependent RNA methyltransferase (SAM MTase) domain. Within a cleft formed at the interface of these two domains, the peptide substrate is thought to bind. The nucleophilic cysteine can be found within a conserved motif, <sup>475</sup>NVCWAK<sup>480</sup>, which differs from that of papain (<sup>22</sup>CGSCWAFS<sup>29</sup>). Mutation of the motif residue, N475, to alanine unexpectedly produced a self-inhibited state in which the N-terminal residues flipped into the substrate-binding cleft. Notably, the N-terminal segment was not hydrolyzed-consistent with a catalytically incompetent state. The N475A mutation resulted in a 70-fold decrease in k<sub>cat</sub>/K<sub>m</sub>. A side chain-substrate interaction was predicted by the structure; the S701A mutation led to a 17-fold increase in K<sub>m</sub>. An Asn at the n-2 position relative to the Cys was also found in the coronaviral papain-like proteases/deubiquitinases (PLpro) of the SARS and MERS viruses, and in several papain-like human ubiquitin specific proteases (USP). The large conformational change in the N475A variant suggests that Asn-475 plays an important role in stabilizing the N-terminal residues and in orienting the carbonyl during nucleophilic attack but does not directly hydrogen bond the oxyanion. The state trapped in crystallo is an unusual result of site-directed mutagenesis but reveals the role of this highly conserved Asn and identifies key substrate-binding contacts that may be exploited by peptide-like inhibitors.</div></front></TEI><affiliations><list><country><li>États-Unis</li></country></list><tree><country name="États-Unis"><noRegion><name sortKey="Compton, Jaimee R" sort="Compton, Jaimee R" uniqKey="Compton J" first="Jaimee R" last="Compton">Jaimee R. Compton</name></noRegion><name sortKey="Hu, Xin" sort="Hu, Xin" uniqKey="Hu X" first="Xin" last="Hu">Xin Hu</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 J" sort="Marugan, Juan J" uniqKey="Marugan J" first="Juan J" last="Marugan">Juan J. Marugan</name><name sortKey="Mickey, Matthew J" sort="Mickey, Matthew J" uniqKey="Mickey M" first="Matthew J" last="Mickey">Matthew J. Mickey</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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