Mutation of Asn-475 in the Venezuelan Equine Encephalitis Virus nsP2 Cysteine Protease Leads to a Self-Inhibited State.
Identifieur interne : 000A69 ( PubMed/Corpus ); précédent : 000A68; suivant : 000A70Mutation of Asn-475 in the Venezuelan Equine Encephalitis Virus nsP2 Cysteine Protease Leads to a Self-Inhibited State.
Auteurs : Jaimee R. Compton ; Matthew J. Mickey ; Xin Hu ; Juan J. Marugan ; Patricia M. LeglerSource :
- Biochemistry [ 1520-4995 ] ; 2017.
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
Links to Exploration step
pubmed:29064679Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Mutation of Asn-475 in the Venezuelan Equine Encephalitis Virus nsP2 Cysteine Protease Leads to a Self-Inhibited State.</title><author><name sortKey="Compton, Jaimee R" sort="Compton, Jaimee R" uniqKey="Compton J" first="Jaimee R" last="Compton">Jaimee R. Compton</name><affiliation><nlm:affiliation>U.S. Naval Research Laboratory , 4555 Overlook Avenue, Washington, D.C. 20375, United States.</nlm:affiliation></affiliation></author><author><name sortKey="Mickey, Matthew J" sort="Mickey, Matthew J" uniqKey="Mickey M" first="Matthew J" last="Mickey">Matthew J. Mickey</name><affiliation><nlm:affiliation>U.S. Naval Academy , Annapolis, Maryland 21402, United States.</nlm:affiliation></affiliation></author><author><name sortKey="Hu, Xin" sort="Hu, Xin" uniqKey="Hu X" first="Xin" last="Hu">Xin Hu</name><affiliation><nlm:affiliation>National Center for Advancing Translational Sciences, National Institutes of Health , Rockville, Maryland 20850, United States.</nlm:affiliation></affiliation></author><author><name sortKey="Marugan, Juan J" sort="Marugan, Juan J" uniqKey="Marugan J" first="Juan J" last="Marugan">Juan J. Marugan</name><affiliation><nlm:affiliation>National Center for Advancing Translational Sciences, National Institutes of Health , Rockville, Maryland 20850, United States.</nlm:affiliation></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><nlm:affiliation>U.S. Naval Research Laboratory , 4555 Overlook Avenue, Washington, D.C. 20375, United States.</nlm:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2017">2017</date><idno type="RBID">pubmed:29064679</idno><idno type="pmid">29064679</idno><idno type="doi">10.1021/acs.biochem.7b00746</idno><idno type="wicri:Area/PubMed/Corpus">000A69</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Corpus" wicri:corpus="PubMed">000A69</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">Mutation of Asn-475 in the Venezuelan Equine Encephalitis Virus nsP2 Cysteine Protease Leads to a Self-Inhibited State.</title><author><name sortKey="Compton, Jaimee R" sort="Compton, Jaimee R" uniqKey="Compton J" first="Jaimee R" last="Compton">Jaimee R. Compton</name><affiliation><nlm:affiliation>U.S. Naval Research Laboratory , 4555 Overlook Avenue, Washington, D.C. 20375, United States.</nlm:affiliation></affiliation></author><author><name sortKey="Mickey, Matthew J" sort="Mickey, Matthew J" uniqKey="Mickey M" first="Matthew J" last="Mickey">Matthew J. Mickey</name><affiliation><nlm:affiliation>U.S. Naval Academy , Annapolis, Maryland 21402, United States.</nlm:affiliation></affiliation></author><author><name sortKey="Hu, Xin" sort="Hu, Xin" uniqKey="Hu X" first="Xin" last="Hu">Xin Hu</name><affiliation><nlm:affiliation>National Center for Advancing Translational Sciences, National Institutes of Health , Rockville, Maryland 20850, United States.</nlm:affiliation></affiliation></author><author><name sortKey="Marugan, Juan J" sort="Marugan, Juan J" uniqKey="Marugan J" first="Juan J" last="Marugan">Juan J. Marugan</name><affiliation><nlm:affiliation>National Center for Advancing Translational Sciences, National Institutes of Health , Rockville, Maryland 20850, United States.</nlm:affiliation></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><nlm:affiliation>U.S. Naval Research Laboratory , 4555 Overlook Avenue, Washington, D.C. 20375, United States.</nlm:affiliation></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="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="enzymology" xml:lang="en"><term>Encephalitis Virus, Venezuelan Equine</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></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><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">29064679</PMID><DateCompleted><Year>2017</Year><Month>12</Month><Day>08</Day></DateCompleted><DateRevised><Year>2018</Year><Month>11</Month><Day>05</Day></DateRevised><Article PubModel="Print-Electronic"><Journal><ISSN IssnType="Electronic">1520-4995</ISSN><JournalIssue CitedMedium="Internet"><Volume>56</Volume><Issue>47</Issue><PubDate><Year>2017</Year><Month>11</Month><Day>28</Day></PubDate></JournalIssue><Title>Biochemistry</Title><ISOAbbreviation>Biochemistry</ISOAbbreviation></Journal><ArticleTitle>Mutation of Asn-475 in the Venezuelan Equine Encephalitis Virus nsP2 Cysteine Protease Leads to a Self-Inhibited State.</ArticleTitle><Pagination><MedlinePgn>6221-6230</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.1021/acs.biochem.7b00746</ELocationID><Abstract><AbstractText>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.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Compton</LastName><ForeName>Jaimee R</ForeName><Initials>JR</Initials><AffiliationInfo><Affiliation>U.S. Naval Research Laboratory , 4555 Overlook Avenue, Washington, D.C. 20375, United States.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Mickey</LastName><ForeName>Matthew J</ForeName><Initials>MJ</Initials><AffiliationInfo><Affiliation>U.S. Naval Academy , Annapolis, Maryland 21402, United States.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Hu</LastName><ForeName>Xin</ForeName><Initials>X</Initials><AffiliationInfo><Affiliation>National Center for Advancing Translational Sciences, National Institutes of Health , Rockville, Maryland 20850, United States.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Marugan</LastName><ForeName>Juan J</ForeName><Initials>JJ</Initials><AffiliationInfo><Affiliation>National Center for Advancing Translational Sciences, National Institutes of Health , Rockville, Maryland 20850, United States.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Legler</LastName><ForeName>Patricia M</ForeName><Initials>PM</Initials><Identifier Source="ORCID">0000-0003-1196-1061</Identifier><AffiliationInfo><Affiliation>U.S. Naval Research Laboratory , 4555 Overlook Avenue, Washington, D.C. 20375, United States.</Affiliation></AffiliationInfo></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013486">Research Support, U.S. Gov't, Non-P.H.S.</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2017</Year><Month>11</Month><Day>09</Day></ArticleDate></Article><MedlineJournalInfo><Country>United States</Country><MedlineTA>Biochemistry</MedlineTA><NlmUniqueID>0370623</NlmUniqueID><ISSNLinking>0006-2960</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D014764">Viral Proteins</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 3.4.22.-</RegistryNumber><NameOfSubstance UI="D003546">Cysteine Endopeptidases</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 3.4.22.-</RegistryNumber><NameOfSubstance UI="C078306">nsP2 proteinase</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D000595" MajorTopicYN="N">Amino Acid Sequence</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D001665" MajorTopicYN="N">Binding Sites</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D001667" MajorTopicYN="N">Binding, Competitive</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D020134" MajorTopicYN="N">Catalytic Domain</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D018360" MajorTopicYN="N">Crystallography, X-Ray</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D003546" MajorTopicYN="N">Cysteine Endopeptidases</DescriptorName><QualifierName UI="Q000737" MajorTopicYN="Y">chemistry</QualifierName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D004666" MajorTopicYN="N">Encephalitis Virus, Venezuelan Equine</DescriptorName><QualifierName UI="Q000201" MajorTopicYN="Y">enzymology</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D025461" MajorTopicYN="Y">Feedback, Physiological</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D006801" 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