Structure of Main Protease from Human Coronavirus NL63: Insights for Wide Spectrum Anti-Coronavirus Drug Design
Identifieur interne : 000327 ( Pmc/Corpus ); précédent : 000326; suivant : 000328Structure of Main Protease from Human Coronavirus NL63: Insights for Wide Spectrum Anti-Coronavirus Drug Design
Auteurs : Fenghua Wang ; Cheng Chen ; Wenjie Tan ; Kailin Yang ; Haitao YangSource :
- Scientific Reports [ 2045-2322 ] ; 2016.
Abstract
First identified in The Netherlands in 2004, human coronavirus NL63 (HCoV-NL63) was found to cause worldwide infections. Patients infected by HCoV-NL63 are typically young children with upper and lower respiratory tract infection, presenting with symptoms including croup, bronchiolitis and pneumonia. Unfortunately, there are currently no effective antiviral therapy to contain HCoV-NL63 infection. CoV genomes encode an integral viral component, main protease (Mpro), which is essential for viral replication through proteolytic processing of RNA replicase machinery. Due to the sequence and structural conservation among all CoVs, Mpro has been recognized as an attractive molecular target for rational anti-CoV drug design. Here we present the crystal structure of HCoV-NL63 Mpro in complex with a Michael acceptor inhibitor N3. Structural analysis, consistent with biochemical inhibition results, reveals the molecular mechanism of enzyme inhibition at the highly conservative substrate-recognition pocket. We show such molecular target remains unchanged across 30 clinical isolates of HCoV-NL63 strains. Through comparative study with Mpros from other human CoVs (including the deadly SARS-CoV and MERS-CoV) and their related zoonotic CoVs, our structure of HCoV-NL63 Mpro provides critical insight into rational development of wide spectrum antiviral therapeutics to treat infections caused by human CoVs.
The online version of this article (doi:10.1038/srep22677) contains supplementary material, which is available to authorized users.
Url:
DOI: 10.1038/srep22677
PubMed: 26948040
PubMed Central: 4780191
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PMC:4780191Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Structure of Main Protease from Human Coronavirus NL63: Insights for Wide Spectrum Anti-Coronavirus Drug Design</title><author><name sortKey="Wang, Fenghua" sort="Wang, Fenghua" uniqKey="Wang F" first="Fenghua" last="Wang">Fenghua Wang</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.33763.32</institution-id><institution-id institution-id-type="ISNI">0000 0004 1761 2484</institution-id><institution>School of Life Sciences, Tianjin University,</institution></institution-wrap>Tianjin, 300072 China</nlm:aff></affiliation></author><author><name sortKey="Chen, Cheng" sort="Chen, Cheng" uniqKey="Chen C" first="Cheng" last="Chen">Cheng Chen</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.33763.32</institution-id><institution-id institution-id-type="ISNI">0000 0004 1761 2484</institution-id><institution>School of Life Sciences, Tianjin University,</institution></institution-wrap>Tianjin, 300072 China</nlm:aff></affiliation><affiliation><nlm:aff id="Aff2"><institution-wrap><institution-id institution-id-type="GRID">grid.488175.7</institution-id><institution>Tianjin International Joint Academy of Biotechnology and Medicine,</institution></institution-wrap>Tianjin, 300457 China</nlm:aff></affiliation></author><author><name sortKey="Tan, Wenjie" sort="Tan, Wenjie" uniqKey="Tan W" first="Wenjie" last="Tan">Wenjie Tan</name><affiliation><nlm:aff id="Aff3">Key Laboratory of Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, 102206 China</nlm:aff></affiliation></author><author><name sortKey="Yang, Kailin" sort="Yang, Kailin" uniqKey="Yang K" first="Kailin" last="Yang">Kailin Yang</name><affiliation><nlm:aff id="Aff4"><institution-wrap><institution-id institution-id-type="GRID">grid.254293.b</institution-id><institution-id institution-id-type="ISNI">0000 0004 0435 0569</institution-id><institution>Cleveland Clinic Lerner College of Medicine of Case Western Reserve University,</institution></institution-wrap>Cleveland, 44195 OH USA</nlm:aff></affiliation></author><author><name sortKey="Yang, Haitao" sort="Yang, Haitao" uniqKey="Yang H" first="Haitao" last="Yang">Haitao Yang</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.33763.32</institution-id><institution-id institution-id-type="ISNI">0000 0004 1761 2484</institution-id><institution>School of Life Sciences, Tianjin University,</institution></institution-wrap>Tianjin, 300072 China</nlm:aff></affiliation><affiliation><nlm:aff id="Aff2"><institution-wrap><institution-id institution-id-type="GRID">grid.488175.7</institution-id><institution>Tianjin International Joint Academy of Biotechnology and Medicine,</institution></institution-wrap>Tianjin, 300457 China</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">26948040</idno><idno type="pmc">4780191</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4780191</idno><idno type="RBID">PMC:4780191</idno><idno type="doi">10.1038/srep22677</idno><date when="2016">2016</date><idno type="wicri:Area/Pmc/Corpus">000327</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000327</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Structure of Main Protease from Human Coronavirus NL63: Insights for Wide Spectrum Anti-Coronavirus Drug Design</title><author><name sortKey="Wang, Fenghua" sort="Wang, Fenghua" uniqKey="Wang F" first="Fenghua" last="Wang">Fenghua Wang</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.33763.32</institution-id><institution-id institution-id-type="ISNI">0000 0004 1761 2484</institution-id><institution>School of Life Sciences, Tianjin University,</institution></institution-wrap>Tianjin, 300072 China</nlm:aff></affiliation></author><author><name sortKey="Chen, Cheng" sort="Chen, Cheng" uniqKey="Chen C" first="Cheng" last="Chen">Cheng Chen</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.33763.32</institution-id><institution-id institution-id-type="ISNI">0000 0004 1761 2484</institution-id><institution>School of Life Sciences, Tianjin University,</institution></institution-wrap>Tianjin, 300072 China</nlm:aff></affiliation><affiliation><nlm:aff id="Aff2"><institution-wrap><institution-id institution-id-type="GRID">grid.488175.7</institution-id><institution>Tianjin International Joint Academy of Biotechnology and Medicine,</institution></institution-wrap>Tianjin, 300457 China</nlm:aff></affiliation></author><author><name sortKey="Tan, Wenjie" sort="Tan, Wenjie" uniqKey="Tan W" first="Wenjie" last="Tan">Wenjie Tan</name><affiliation><nlm:aff id="Aff3">Key Laboratory of Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, 102206 China</nlm:aff></affiliation></author><author><name sortKey="Yang, Kailin" sort="Yang, Kailin" uniqKey="Yang K" first="Kailin" last="Yang">Kailin Yang</name><affiliation><nlm:aff id="Aff4"><institution-wrap><institution-id institution-id-type="GRID">grid.254293.b</institution-id><institution-id institution-id-type="ISNI">0000 0004 0435 0569</institution-id><institution>Cleveland Clinic Lerner College of Medicine of Case Western Reserve University,</institution></institution-wrap>Cleveland, 44195 OH USA</nlm:aff></affiliation></author><author><name sortKey="Yang, Haitao" sort="Yang, Haitao" uniqKey="Yang H" first="Haitao" last="Yang">Haitao Yang</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.33763.32</institution-id><institution-id institution-id-type="ISNI">0000 0004 1761 2484</institution-id><institution>School of Life Sciences, Tianjin University,</institution></institution-wrap>Tianjin, 300072 China</nlm:aff></affiliation><affiliation><nlm:aff id="Aff2"><institution-wrap><institution-id institution-id-type="GRID">grid.488175.7</institution-id><institution>Tianjin International Joint Academy of Biotechnology and Medicine,</institution></institution-wrap>Tianjin, 300457 China</nlm:aff></affiliation></author></analytic><series><title level="j">Scientific Reports</title><idno type="eISSN">2045-2322</idno><imprint><date when="2016">2016</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>First identified in The Netherlands in 2004, human coronavirus NL63 (HCoV-NL63) was found to cause worldwide infections. Patients infected by HCoV-NL63 are typically young children with upper and lower respiratory tract infection, presenting with symptoms including croup, bronchiolitis and pneumonia. Unfortunately, there are currently no effective antiviral therapy to contain HCoV-NL63 infection. CoV genomes encode an integral viral component, main protease (M<sup>pro</sup>), which is essential for viral replication through proteolytic processing of RNA replicase machinery. Due to the sequence and structural conservation among all CoVs, M<sup>pro</sup> has been recognized as an attractive molecular target for rational anti-CoV drug design. Here we present the crystal structure of HCoV-NL63 M<sup>pro</sup> in complex with a Michael acceptor inhibitor N3. Structural analysis, consistent with biochemical inhibition results, reveals the molecular mechanism of enzyme inhibition at the highly conservative substrate-recognition pocket. We show such molecular target remains unchanged across 30 clinical isolates of HCoV-NL63 strains. Through comparative study with M<sup>pro</sup>s from other human CoVs (including the deadly SARS-CoV and MERS-CoV) and their related zoonotic CoVs, our structure of HCoV-NL63 M<sup>pro</sup> provides critical insight into rational development of wide spectrum antiviral therapeutics to treat infections caused by human CoVs.</p><sec><title>Electronic supplementary material</title><p>The online version of this article (doi:10.1038/srep22677) contains supplementary material, which is available to authorized users.</p></sec></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Weiss, Sr" uniqKey="Weiss S">SR Weiss</name></author><author><name sortKey="Navas Martin, S" uniqKey="Navas Martin S">S Navas-Martin</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Masters, Ps" uniqKey="Masters P">PS Masters</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Siddell, S" uniqKey="Siddell 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Sci Rep</journal-id><journal-id journal-id-type="iso-abbrev">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type="epub">2045-2322</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">26948040</article-id><article-id pub-id-type="pmc">4780191</article-id><article-id pub-id-type="publisher-id">BFsrep22677</article-id><article-id pub-id-type="doi">10.1038/srep22677</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Structure of Main Protease from Human Coronavirus NL63: Insights for Wide Spectrum Anti-Coronavirus Drug Design</article-title></title-group><contrib-group><contrib contrib-type="author" equal-contrib="yes"><name><surname>Wang</surname><given-names>Fenghua</given-names></name><xref ref-type="aff" rid="Aff1">1</xref></contrib><contrib contrib-type="author" equal-contrib="yes"><name><surname>Chen</surname><given-names>Cheng</given-names></name><xref ref-type="aff" rid="Aff1">1</xref><xref ref-type="aff" rid="Aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Tan</surname><given-names>Wenjie</given-names></name><xref ref-type="aff" rid="Aff3">3</xref></contrib><contrib contrib-type="author"><name><surname>Yang</surname><given-names>Kailin</given-names></name><address><email>yangk2@ccf.org</email></address><xref ref-type="aff" rid="Aff4">4</xref></contrib><contrib contrib-type="author"><name><surname>Yang</surname><given-names>Haitao</given-names></name><address><email>yanght@tju.edu.cn</email></address><xref ref-type="aff" rid="Aff1">1</xref><xref ref-type="aff" rid="Aff2">2</xref></contrib><aff id="Aff1"><label>1</label><institution-wrap><institution-id institution-id-type="GRID">grid.33763.32</institution-id><institution-id institution-id-type="ISNI">0000 0004 1761 2484</institution-id><institution>School of Life Sciences, Tianjin University,</institution></institution-wrap>Tianjin, 300072 China</aff><aff id="Aff2"><label>2</label><institution-wrap><institution-id institution-id-type="GRID">grid.488175.7</institution-id><institution>Tianjin International Joint Academy of Biotechnology and Medicine,</institution></institution-wrap>Tianjin, 300457 China</aff><aff id="Aff3"><label>3</label>Key Laboratory of Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, 102206 China</aff><aff id="Aff4"><label>4</label><institution-wrap><institution-id institution-id-type="GRID">grid.254293.b</institution-id><institution-id institution-id-type="ISNI">0000 0004 0435 0569</institution-id><institution>Cleveland Clinic Lerner College of Medicine of Case Western Reserve University,</institution></institution-wrap>Cleveland, 44195 OH USA</aff></contrib-group><pub-date pub-type="epub"><day>7</day><month>3</month><year>2016</year></pub-date><pub-date pub-type="pmc-release"><day>7</day><month>3</month><year>2016</year></pub-date><pub-date pub-type="collection"><year>2016</year></pub-date><volume>6</volume><elocation-id>22677</elocation-id><history><date date-type="received"><day>4</day><month>12</month><year>2015</year></date><date date-type="accepted"><day>17</day><month>2</month><year>2016</year></date></history><permissions><copyright-statement>© The Author(s) 2016</copyright-statement><license license-type="OpenAccess"><license-p>This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">http://creativecommons.org/licenses/by/4.0/</ext-link></license-p></license></permissions><abstract id="Abs1"><p>First identified in The Netherlands in 2004, human coronavirus NL63 (HCoV-NL63) was found to cause worldwide infections. Patients infected by HCoV-NL63 are typically young children with upper and lower respiratory tract infection, presenting with symptoms including croup, bronchiolitis and pneumonia. Unfortunately, there are currently no effective antiviral therapy to contain HCoV-NL63 infection. CoV genomes encode an integral viral component, main protease (M<sup>pro</sup>), which is essential for viral replication through proteolytic processing of RNA replicase machinery. Due to the sequence and structural conservation among all CoVs, M<sup>pro</sup> has been recognized as an attractive molecular target for rational anti-CoV drug design. Here we present the crystal structure of HCoV-NL63 M<sup>pro</sup> in complex with a Michael acceptor inhibitor N3. Structural analysis, consistent with biochemical inhibition results, reveals the molecular mechanism of enzyme inhibition at the highly conservative substrate-recognition pocket. We show such molecular target remains unchanged across 30 clinical isolates of HCoV-NL63 strains. Through comparative study with M<sup>pro</sup>s from other human CoVs (including the deadly SARS-CoV and MERS-CoV) and their related zoonotic CoVs, our structure of HCoV-NL63 M<sup>pro</sup> provides critical insight into rational development of wide spectrum antiviral therapeutics to treat infections caused by human CoVs.</p><sec><title>Electronic supplementary material</title><p>The online version of this article (doi:10.1038/srep22677) contains supplementary material, which is available to authorized users.</p></sec></abstract><kwd-group kwd-group-type="npg-subject"><title>Subject terms</title><kwd>Virology</kwd><kwd>X-ray crystallography</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2016</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="Sec1"><title>Introduction</title><p>Coronaviruses (CoVs) are a diverse group of enveloped positive-strand RNA viruses in the family <italic>Coronaviridae</italic><sup><xref ref-type="bibr" rid="CR1">1</xref>,<xref ref-type="bibr" rid="CR2">2</xref></sup>. CoVs have been identified in a wide variety of hosts, including mammals and birds and are shown to cause a number of respiratory and enteric diseases<sup><xref ref-type="bibr" rid="CR1">1</xref>,<xref ref-type="bibr" rid="CR3">3</xref>,<xref ref-type="bibr" rid="CR4">4</xref></sup>. In 2003, the global epidemic of an atypical form of pneumonia named severe acute respiratory syndrome (SARS) led to the discovery of SARS-CoV, a previously unknown CoV, as the etiologic pathogen<sup><xref ref-type="bibr" rid="CR5">5</xref>,<xref ref-type="bibr" rid="CR6">6</xref>,<xref ref-type="bibr" rid="CR7">7</xref>,<xref ref-type="bibr" rid="CR8">8</xref></sup>. Started in South China, SARS outbreak quickly resulted in more than 800 deaths worldwide<sup><xref ref-type="bibr" rid="CR9">9</xref></sup>. Patients with SARS-CoV infection developed diffuse alveolar damage with the potential to progress into acute respiratory distress syndrome and eventually death<sup><xref ref-type="bibr" rid="CR10">10</xref></sup>. Almost 10 years later, another previously unknown CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), was found to cause a new epidemic starting in the Arabian Peninsula in 2012<sup><xref ref-type="bibr" rid="CR11">11</xref>,<xref ref-type="bibr" rid="CR12">12</xref>,<xref ref-type="bibr" rid="CR13">13</xref></sup>. MERS infection led to acute pneumonia and renal failure, with mortality rate as high as 50% in hospitalized patients<sup><xref ref-type="bibr" rid="CR14">14</xref>,<xref ref-type="bibr" rid="CR15">15</xref></sup>. In addition to the deadly SARS-CoV and MERS-CoV, 4 other human CoVs have been identified so far, namely HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1, which are known to cause comparatively mild common colds<sup><xref ref-type="bibr" rid="CR9">9</xref>,<xref ref-type="bibr" rid="CR16">16</xref>,<xref ref-type="bibr" rid="CR17">17</xref></sup>. According to their genomic sequences, these 6 HCoVs are further classified into <italic>alphacoronavirus</italic> genus (HCoV-229E and HCoV-NL63) and <italic>betacoronavirus</italic> genus (HCoV-OC43, HCoV-HKU1, SARS-CoV and MERS-CoV)<sup><xref ref-type="bibr" rid="CR12">12</xref>,<xref ref-type="bibr" rid="CR18">18</xref></sup>. The emergence of CoV infection in human beings are believed to begin with zoonotic transmission from animal reservoirs<sup><xref ref-type="bibr" rid="CR9">9</xref></sup>. For example, high degree of genomic sequence similarity was shown between bovine CoV and HCoV-OC43, suggesting a relatively recent animal-to-human transmission<sup><xref ref-type="bibr" rid="CR11">11</xref>,<xref ref-type="bibr" rid="CR19">19</xref></sup>. In the case of human SARS-CoV, recent studies identified several SARS-like bat CoVs with over 95% genomic sequence identity, suggesting bats as the potential zoonotic reservoir<sup><xref ref-type="bibr" rid="CR20">20</xref>,<xref ref-type="bibr" rid="CR21">21</xref>,<xref ref-type="bibr" rid="CR22">22</xref></sup>. While dromedary camels are suspected to be either reservoir or vector for MERS, as genomic sequence of isolated dromedary MERS-CoV was found identical to that of human MERS-CoV<sup><xref ref-type="bibr" rid="CR23">23</xref>,<xref ref-type="bibr" rid="CR24">24</xref>,<xref ref-type="bibr" rid="CR25">25</xref></sup>.</p><p>Human coronavirus NL63 (HCoV-NL63) was first isolated in 2004 from a 7-month-old child suffering from bronchiolitis and conjunctivitis in the Netherlands<sup><xref ref-type="bibr" rid="CR16">16</xref></sup>. HCoV-NL63 has been documented to circulate in human population worldwide<sup><xref ref-type="bibr" rid="CR26">26</xref>,<xref ref-type="bibr" rid="CR27">27</xref>,<xref ref-type="bibr" rid="CR28">28</xref>,<xref ref-type="bibr" rid="CR29">29</xref>,<xref ref-type="bibr" rid="CR30">30</xref>,<xref ref-type="bibr" rid="CR31">31</xref>,<xref ref-type="bibr" rid="CR32">32</xref>,<xref ref-type="bibr" rid="CR33">33</xref></sup> and is considered the causative pathogen for up to 10% of all respiratory illnesses<sup><xref ref-type="bibr" rid="CR34">34</xref>,<xref ref-type="bibr" rid="CR35">35</xref>,<xref ref-type="bibr" rid="CR36">36</xref>,<xref ref-type="bibr" rid="CR37">37</xref></sup>. Infected patients are typically young children with upper and lower respiratory tract infection, presenting with symptoms including croup, bronchiolitis and pneumonia<sup><xref ref-type="bibr" rid="CR38">38</xref>,<xref ref-type="bibr" rid="CR39">39</xref></sup>. Nevertheless, infections in adults have also been reported, though consequences could be more severe in those with compromised immune system or other comorbidities<sup><xref ref-type="bibr" rid="CR40">40</xref>,<xref ref-type="bibr" rid="CR41">41</xref>,<xref ref-type="bibr" rid="CR42">42</xref></sup>. Similar to SARS-CoV, HCoV-NL63 also uses angiotensin-converting enzyme 2 (ACE2) as the receptor for cellular entry<sup><xref ref-type="bibr" rid="CR43">43</xref></sup>. Full genome sequences of HCoV-NL63 have been determined, revealing a mosaic structure with multiple recombination sites which indicate that possible mutation and recombination could occur when co-infected with other CoVs<sup><xref ref-type="bibr" rid="CR44">44</xref>,<xref ref-type="bibr" rid="CR45">45</xref></sup>. Based on molecular clock analysis, HCoV-NL63 shares common ancestry with bat <italic>alphacoronavirus</italic> sequences, with probable divergence 563–822 years ago<sup><xref ref-type="bibr" rid="CR46">46</xref></sup>. However, the direct bat ancestor of HCoV-NL63 has not been found yet<sup><xref ref-type="bibr" rid="CR47">47</xref></sup>.</p><p>Currently there are no approved antiviral drugs or vaccines against human CoV infection, though several compounds have been investigated in pre-clinical studies<sup><xref ref-type="bibr" rid="CR9">9</xref></sup>. From a public health perspective, no effective antiviral strategy is available in face of future CoV emergence, potentially transmitted from the vast and mutable zoonotic reservoir. Previously, we have demonstrated that main protease (M<sup>pro</sup>) is a conserved drug target throughout the subfamily <italic>Coronavirinae</italic>, which is suitable for designing wide-spectrum inhibitors<sup><xref ref-type="bibr" rid="CR48">48</xref>,<xref ref-type="bibr" rid="CR49">49</xref></sup>. The 5′ two-thirds of coronaviral genome is consisted of open reading frame 1 (ORF1), which encodes two large polypeptides of the replicase machinery: pp1a and through ribosomal frameshift, pp1ab<sup><xref ref-type="bibr" rid="CR18">18</xref></sup>. These two polypeptides are cotranslationally cleaved into mature nonstructural proteins (Nsps) through two proteases encoded in the 5′ region of ORF1: papain-like protease (PLP) and 3C-like protease (3CL or Nsp5)<sup><xref ref-type="bibr" rid="CR50">50</xref>,<xref ref-type="bibr" rid="CR51">51</xref></sup>. 3CL protease is more commonly known as M<sup>pro</sup> because of its dominant role in the posttranslational processing of the replicase polyprotein. The M<sup>pro</sup>s from different human and animal CoVs are known to share significant homology in both primary amino acid sequence and 3D architecture, providing a strong structural basis for designing wide-spectrum anti-CoV inhibitors<sup><xref ref-type="bibr" rid="CR48">48</xref>,<xref ref-type="bibr" rid="CR49">49</xref>,<xref ref-type="bibr" rid="CR52">52</xref>,<xref ref-type="bibr" rid="CR53">53</xref>,<xref ref-type="bibr" rid="CR54">54</xref>,<xref ref-type="bibr" rid="CR55">55</xref></sup>. They employ a similar substrate-binding pocket, usually with a requirement for glutamine at P1 position and a preference for leucine/methionine at P2 position. Interestingly, in contrast to other HCoVs, only HCoV-NL63 and HCoV-HKU1 exhibit a unique substrate preference of histidine in P1 position at the cleavage site between nsp13 and nsp14<sup><xref ref-type="bibr" rid="CR37">37</xref>,<xref ref-type="bibr" rid="CR53">53</xref></sup>. The structural and pharmaceutical significance of the P1 position preference of HCoV-NL63 M<sup>pro</sup> remains to be addressed.</p><p>Here, we report the crystal structure of HCoV-NL63 M<sup>pro</sup> in complex with a synthetic peptidomimetic inhibitor, N3. Structural analysis reveals relative conservation at the P1 pocket. Through comparison with M<sup>pro</sup>s from other CoVs, we provide structural insight into rational drug design at a conserved target across pathological human coronaviruses and their related zoonotic counterparts.</p></sec><sec id="Sec2"><title>Results</title><sec id="Sec3"><title>Structural Overview</title><p>There are two protein molecules in an asymmetric unit. The two molecules form a typical homodimer (<xref rid="Fig1" ref-type="fig">Fig. 1a</xref>), which has been observed in the crystal structures of other CoV M<sup>pro</sup>s<sup><xref ref-type="bibr" rid="CR48">48</xref>,<xref ref-type="bibr" rid="CR52">52</xref>,<xref ref-type="bibr" rid="CR53">53</xref></sup>. Previous studies have demonstrated the existence of M<sup>pro</sup> homodimer in solution which is also the only active form of the enzyme<sup><xref ref-type="bibr" rid="CR56">56</xref>,<xref ref-type="bibr" rid="CR57">57</xref>,<xref ref-type="bibr" rid="CR58">58</xref>,<xref ref-type="bibr" rid="CR59">59</xref></sup>, supporting the physiological relevance of structural findings. A structural comparison of protomer A in M<sup>pro</sup>-N3 complex with that in apo enzyme (PDB ID: 3TLO; C. P. Chuck & K. B. Wong, unpublished work) revealed an overall architecture of three domains (<xref rid="Fig1" ref-type="fig">Fig. 1b</xref>) in each protomer, a common feature among CoV M<sup>pro</sup> structures. Domain I (residues 8–100) and domain II (residues 101–183) together form a chymotrypsin-like fold and the substrate-binding site is located in a cleft formed between domain I and domain II. The catalytic dyad composed of Cys144 and His41 lies in the center of substrate-binding site. Domain III (residues 200–303) of HCoV-NL63 M<sup>pro</sup> is composed of a globular antiparallel α-helical cluster, a unique feature of CoV M<sup>pro</sup> that is required for homodimer formation. Domain III is connected to domain II through a long loop region of 16 residues. X-ray data-processing and refinement statistics are included in <xref rid="Tab1" ref-type="table">Table 1</xref>.<table-wrap id="Tab1"><label>Table 1</label><caption><p>X-ray data-processing and refinement statistics.</p></caption><table frame="hsides" rules="groups"><thead><tr><th>Statistics</th><th align="center">Value for the HCoV-NL63 M<sup>pro</sup>-N3 complex</th></tr></thead><tbody><tr><td align="left" colspan="2">Data collection</td></tr><tr><td> Wavelength (Å)</td><td>1.0000</td></tr><tr><td> Resolution limit (Å)</td><td>50.0–2.85 (2.90–2.85)</td></tr><tr><td> Space group</td><td><italic>P</italic>4<sub>1</sub>2<sub>1</sub>2</td></tr><tr><td align="left" colspan="2">Cell parameters</td></tr><tr><td> a (Å)</td><td>87.2</td></tr><tr><td> b (Å)</td><td>87.2</td></tr><tr><td> c (Å)</td><td>212.1</td></tr><tr><td> α = β = γ (°)</td><td>90</td></tr><tr><td>Total no. of reflections</td><td>283898</td></tr><tr><td>No. of unique reflections</td><td>19967</td></tr><tr><td>Completeness (%)</td><td>100 (100)</td></tr><tr><td>Redundancy</td><td>14.2 (12.6)</td></tr><tr><td>R<sub>merge</sub> (%)</td><td>14.4 (46.6)</td></tr><tr><td>Sigma cutoff</td><td>0</td></tr><tr><td><italic>I/σ(I)</italic></td><td>21.5 (6.1)</td></tr><tr><td align="left" colspan="2">Refinement</td></tr><tr><td> Resolution range (Å)</td><td>50.0–2.85</td></tr><tr><td> R<sub>work</sub> (%)</td><td>19.3</td></tr><tr><td> R<sub>free</sub> (%)</td><td>24.1</td></tr><tr><td align="left" colspan="2">Rmsd from ideal geometry</td></tr><tr><td> Bonds (Å)</td><td>0.007</td></tr><tr><td> Angles (°)</td><td>1.16</td></tr><tr><td>Avg B factor (Å<sup>2</sup>)</td><td>27.6</td></tr><tr><td> Protein</td><td>28.1</td></tr><tr><td> Small molecule</td><td>28.3</td></tr><tr><td align="left" colspan="2">Ramachandran plot</td></tr><tr><td> Favored (%)</td><td>96.0</td></tr><tr><td> Allowed (%)</td><td>4.0</td></tr><tr><td> Outliers (%)</td><td>0.0</td></tr></tbody></table></table-wrap><fig id="Fig1"><label>Figure 1</label><caption><p>Structural overview of HCoV-NL63 M<sup>pro</sup>.</p><p>(<bold>a</bold>) Overview of homodimer in one asymmetric unit (A: slate and B: deep salmon). Protomers are shown in cartoons and N3 inhibitors are shown as green sticks. (<bold>b</bold>) Structural alignment of protomer A in M<sup>pro</sup>-N3 (slate) complex with that in apo enzyme (light orange, PDB ID: 3TLO; C. P. Chuck & K. B. Wong, unpublished work). The backbone is shown in cartoons and N3 inhibitor is presented as green sticks.</p></caption><graphic xlink:href="41598_2016_Article_BFsrep22677_Fig1_HTML" id="d29e896"></graphic></fig></p></sec><sec id="Sec4"><title>Michael acceptor and inhibitor binding at active site</title><p>Michael acceptor inhibitors such as N3 (<xref rid="Fig2" ref-type="fig">Fig. 2a</xref>) undergoes mechanism-based inhibition to achieve covalent irreversible inactivation, as shown in the <xref rid="Equ1" ref-type="">equation (1)</xref>:<fig id="Fig2"><label>Figure 2</label><caption><p>Structure of inhibitor N3 and its interaction with HCoV-NL63 M<sup>pro</sup>.</p><p>(<bold>a)</bold> The structure of compound N3. (<bold>b</bold>) A stereo view of N3 bound to the substrate-binding pocket of HCoV-NL63 M<sup>pro</sup> at 2.85 Å. N3 inhibitor is shown in green and covered by an omit map contoured at 1.0 σ. Residues forming the substrate-binding pocket are shown in silver. <bold>(c)</bold> Detailed view of the interaction between N3 and HCoV-NL63 M<sup>pro</sup>. The N3 inhibitor is shown in green. Hydrogen bonds are shown as dashed lines labeled with interaction distances. The covalent bond between N3 and Sγ of Cys144 is labeled in red.</p></caption><graphic xlink:href="41598_2016_Article_BFsrep22677_Fig2_HTML" id="d29e936"></graphic></fig><disp-formula id="Equ1"><graphic xlink:href="41598_2016_Article_BFsrep22677_Equ1_HTML.gif" position="anchor"></graphic></disp-formula></p><p>The inhibitor first forms a reversible complex (EI) with the enzyme under the equilibrium-binding constant <italic>K</italic><sub><italic>i</italic></sub>. It then undergoes nucleophilic attack by the active site Cys of the enzyme, leading to the formation of a stable covalent bond (E-I). This step is governed by the inactivation rate constant, <italic>k</italic><sub><italic>3</italic></sub>. Using a CoV consensus substrate reported previously<sup><xref ref-type="bibr" rid="CR48">48</xref>,<xref ref-type="bibr" rid="CR49">49</xref>,<xref ref-type="bibr" rid="CR53">53</xref></sup>, we first determined the values for <italic>K</italic><sub><italic>m</italic></sub> and <italic>k</italic><sub><italic>cat</italic></sub> of the apo enzyme of HCoV-NL63 M<sup>pro</sup>, as 50.8 ± 3.4 μM and 0.098 ± 0.004 s<sup>−1</sup>, respectively (<xref rid="Tab2" ref-type="table">Table 2</xref>). Cross comparison with M<sup>pro</sup>s from other human CoVs revealed that the kinetic parameters of HCoV-NL63 M<sup>pro</sup> is relatively close to those for SARS-CoV (<italic>K</italic><sub><italic>m</italic></sub> = 129 ± 7 μM, <italic>k</italic><sub><italic>cat</italic></sub> = 0.14 ± 0.01 s<sup>−1</sup>)<sup><xref ref-type="bibr" rid="CR49">49</xref></sup>. Rather <italic>K</italic><sub><italic>m</italic></sub> of HCoV-NL63 is higher than that for HCoV-229E, while its <italic>k</italic><sub><italic>cat</italic></sub> is approximately ten fold larger than those for HCoV-229E and HCoV-HKU1. We then added inhibitor N3 to the kinetic assay of HCoV-NL63 M<sup>pro</sup> and calculated the <italic>K</italic><sub><italic>i</italic></sub> and <italic>k</italic><sub><italic>3</italic></sub> as 11.3 ± 1.0 μM and 42.4 ± 5.0 (10<sup>−3</sup>∙s<sup>−1</sup>), respectively (<xref rid="Tab2" ref-type="table">Table 2</xref>). Although the <italic>ki</italic> for HCoV-NL63 is higher compared with those for SARS-CoV and HCoV-229E, indicating a lower affinity of inhibitor N3 to the apo enzyme, its <italic>k</italic><sub><italic>3</italic></sub> is significantly larger, which strongly supports the ability of N3 to achieve mechanism-based irreversible inhibition against HCoV-NL63 M<sup>pro</sup>.<table-wrap id="Tab2"><label>Table 2</label><caption><p>Enzyme activity and N3 inhibition data for HCoV-NL63 M<sup>pro</sup>.</p></caption><table frame="hsides" rules="groups"><thead><tr><th rowspan="2">Virus M<sup>pro</sup></th><th rowspan="2"><italic>K</italic><sub><italic>m</italic></sub> (μM)</th><th rowspan="2"><italic>k</italic><sub><italic>cat</italic></sub> (s<sup>−1</sup>)</th><th align="center" colspan="2">Inhibitor N3</th><th rowspan="2">Data Source</th></tr><tr><th><italic>K</italic><sub><italic>i</italic></sub> (μM)</th><th><italic>k</italic><sub><italic>3</italic></sub> (10<sup>−3</sup>∙s<sup>−1</sup>)</th></tr></thead><tbody><tr><td>HCoV-NL63</td><td>50.8 ± 3.4</td><td>0.098 ± 0.004</td><td>11.3 ± 1.0</td><td>42.4 ± 5.0</td><td>This study</td></tr><tr><td>SARS-CoV</td><td>129 ± 7</td><td>0.14 ± 0.01</td><td>9.0 ± 0.8</td><td>3.1 ± 0.5</td><td><xref ref-type="bibr" rid="CR49">49</xref></td></tr><tr><td>HCoV-229E</td><td>29.8 ± 0.9</td><td>1.27 ± 0.09</td><td>1.67 ± 0.18</td><td>18.0 ± 1.1</td><td><xref ref-type="bibr" rid="CR49">49</xref></td></tr><tr><td>HCoV-HKU1</td><td>83.2 ± 13.3</td><td>1.1 ± 0.12</td><td align="center">−</td><td align="center">−</td><td><xref ref-type="bibr" rid="CR53">53</xref></td></tr></tbody></table></table-wrap></p><p>Analysis of the complex structure of HCoV-NL63 M<sup>pro</sup> bound to inhibitor N3 provides further insight into the inhibition mechanism (<xref rid="Fig2" ref-type="fig">Fig. 2b</xref>). Since N3 binds to Protomer A and B similarly, we will only look into the binding mode of N3 in Protomer A below. Cβ of vinyl group on inhibitor N3 forms a standard 1.8 Å covalent bond with the Sγ atom of Cys144, as evidenced by clear electron density (<xref rid="Fig2" ref-type="fig">Fig. 2b</xref>). This indicates that a Michael addition reaction occurred between N3 and the catalytic site Cys144. Compared with the apo enzyme, only one specific conformation is observed due to the formation of covalent bond while there exists a double conformation for Sγ in the apo enzyme. The carbonyl oxygen of ester on N3 is very close to the backbone NH of Gly142 and the backbone of NH of Cys144 (<xref rid="Fig2" ref-type="fig">Fig. 2c</xref>), which mimics the tetrahedral oxyanion intermediate state formed during serine protease cleavage and provides additional support to anchor the Michael acceptor. The benzyl ester part of N3 further extends into the S1’ pocket, forming Van der Waals interaction with Val26 and Leu27 (<xref rid="Fig2" ref-type="fig">Fig. 2b</xref>).</p><p>Comparison between the molecular surfaces of HCoV-NL63 M<sup>pro</sup> complexed with N3 and the apo enzyme reveals that the pocket accommodating N3 undergoes significant conformation changes upon inhibitor binding (<xref rid="Fig3" ref-type="fig">Fig. 3a,b</xref>) as following: (1) the imidazole group of His41 swifts ~5 Å towards the hydrophobic core of the protein to better accommodate P1’ site; (2) the main chain of residues 138–142 which constitute the outer wall of S1 subsite moves toward the lactam ring, causing the shrinking of S1 pocket; (3) residues 45–51 flip over to act as a lid to cover P2 subsite; (4) residues 164–168 and 187–191 extend into opposite directions to host N3. In the following sections, we will describe the detailed structural features of the subpockets.<fig id="Fig3"><label>Figure 3</label><caption><p>Surface representation of native HCoV-NL63 and its complex with inhibitor N3.</p><p>(<bold>a)</bold> Surface representation of substrate-binding pockets from apo enzyme of HCoV-NL63 M<sup>pro</sup> (marine, PDB ID: 3TLO). The S1, S2, S4 and S1’ pockets and residues forming the substrate-binding pocket are labeled. <bold>(b)</bold> Surface representation of HCoV-NL63 M<sup>pro</sup> (marine) in complex with N3 inhibitor (green). Water molecules are shown as red spheres. The P1-P5 and P1’ groups of N3 inhibitor are labeled together with residues forming the substrate-binding pocket.</p></caption><graphic xlink:href="41598_2016_Article_BFsrep22677_Fig3_HTML" id="d29e1268"></graphic></fig></p></sec><sec id="Sec5"><title>Smaller S1 pocket to accommodate P1 histidine</title><p>Coronaviral M<sup>pro</sup>s are known to have strong preference to glutamine (Q) at P1 site of substrate. Genome sequence analysis of HCoV-NL63 revealed that 10 out of 11 M<sup>pro</sup> cleavage sites bear glutamine at P1 position, except that the recognition site between nsp13 (helicase) and nsp14 (exonuclease) where histidine replaces glutamine at P1 site<sup><xref ref-type="bibr" rid="CR37">37</xref></sup>. This feature is unique only in genomes of HCoV-NL63 (NC_005831) and HCoV-HKU1 (NC_006577). In contrast, glutamine is the exclusive residue in P1 among all cleavage sites for the other four human CoVs, including HCoV-229E (NC_002645), HCoV-OC43 (NC_005147), SARS-CoV (NC_004718) and MERS-CoV (JX869059). Such novel substrate specificity at P1 in HCoV-NL63 and HCoV-HKU1 implies unique structural feature of S1 subpocket. The crystal structure of HCoV-NL63 M<sup>pro</sup> shows that the size of its S1 pocket is comparable to that of HCoV-HKU1, but smaller than those of HCoV-229E and SARS-CoV. Clearly, a smaller S1 pocket in HCoV-NL63 could better accommodate the smaller side chain of histidine residue at P1 of nsp13-nsp14 and facilitate cleavage when weakened oxyanion hole is formed.</p><p>In our enzyme-inhibitor complex structure, the lactam ring of N3 molecule serves as a structural analog to glutamine or histidine at P1 position (<xref rid="Fig2" ref-type="fig">Fig. 2a</xref>) and protrudes into the S1 pocket by forming a 2.5 Å hydrogen bond between the lactam oxygen and the imidazole ring NH of His163 (<xref rid="Fig2" ref-type="fig">Fig. 2b,c</xref>), which is similar to those in the complex structures of SARS-CoV M<sup>pro</sup> and HCoV-HKU1 M<sup>pro</sup> with inhibitor N3<sup><xref ref-type="bibr" rid="CR49">49</xref>,<xref ref-type="bibr" rid="CR53">53</xref></sup>. Furthermore, the NH of N3 lactam ring forms two hydrogen bonds with Oε1 of Glu166 (3.1 Å) and the carbonyl oxygen atom of Phe139 (3.2 Å) respectively, which provide additional support to stabilize the lactam ring in S1 pocket. The backbone NH of P1 residue on N3 also forms a 3.0 Å hydrogen bond with the backbone carbonyl oxygen from Gln164, favorably accommodating the S1 pocket.</p></sec><sec id="Sec6"><title>S2 pocket</title><p>The P2 position of natural M<sup>pro</sup> cleavage sites across of the genomes of six human CoVs usually prefers a hydrophobic residue (<xref rid="Tab3" ref-type="table">Table 3</xref>). It is surprising that the P2 residues are completely identical among all 11 cleavage sites between the two alphacoronaviruses, HCoV-NL63 and HCoV-229E. In a majority of cases, it is leucine residue occupying the P2 position, with valine at nsp6-nsp7 and isoleucine at nsp10-nsp11. On the other hand, more variations are observed in the P2 position among betacoronaviruses, with more alternative residues such as methionine, phenylalanine and proline, which might indicate less stringency of P2 specificity. The P2 site of inhibitor N3 mimics the side chain of leucine, in order to cover the maximum spectrum of natural M<sup>pro</sup> substrates for human CoVs<sup><xref ref-type="bibr" rid="CR49">49</xref></sup>. As observed in the HCoV-NL63 structure with N3, the aliphatic isobutyl side chain of P2 protrudes into the deep S2 pocket via interactions with the alkyl portion of the side chains of Asp187, Pro189 and is well accommodated onto the Van der Waals surface of the pocket (<xref rid="Fig2" ref-type="fig">Fig. 2b</xref>).<table-wrap id="Tab3"><label>Table 3</label><caption><p>P2 residues from genomes of six human CoVs.</p></caption><table frame="hsides" rules="groups"><thead><tr><th rowspan="2">No.</th><th align="center" rowspan="2">Cleavage Site</th><th align="center" colspan="2">Alphacoronavirus</th><th align="center" colspan="4">Betacoronavirus</th></tr><tr><th align="center">HCoV-NL63</th><th align="center">HCoV-229E</th><th align="center">HCoV-HKU1</th><th align="center">HCoV-OC43</th><th align="center">SARS-CoV</th><th align="center">MERS-CoV</th></tr></thead><tbody><tr><td>1</td><td>nsp4-nsp5</td><td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td>L</td></tr><tr><td>2</td><td>nsp5-nsp6</td><td>L</td><td>L</td><td>L</td><td>L</td><td>F</td><td>M</td></tr><tr><td>3</td><td>nsp6-nsp7</td><td>V</td><td>V</td><td>I</td><td>F</td><td>V</td><td>M</td></tr><tr><td>4</td><td>nsp7-nsp8</td><td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td>L</td></tr><tr><td>5</td><td>nsp8-nsp9</td><td>L</td><td>L</td><td>M</td><td>L</td><td>L</td><td>L</td></tr><tr><td>6</td><td>nsp9-nsp10</td><td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td>L</td></tr><tr><td>7</td><td>nsp10-nsp11</td><td>I</td><td>I</td><td>V</td><td>V</td><td>M</td><td>P</td></tr><tr><td>8</td><td>nsp12-nsp13</td><td>L</td><td>L</td><td>M</td><td>M</td><td>L</td><td>L</td></tr><tr><td>9</td><td>nsp13-nsp14</td><td>L</td><td>L</td><td>L</td><td>V</td><td>L</td><td>L</td></tr><tr><td>10</td><td>nsp14-nsp15</td><td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td>V</td></tr><tr><td>11</td><td>nsp15-nsp16</td><td>L</td><td>L</td><td>M</td><td>L</td><td>L</td><td>L</td></tr></tbody></table></table-wrap></p><p>To determine the structural diversity in S2 pocket, we then superimposed the backbones of four know M<sup>pro</sup> structures from human CoVs. The lid of the S2 pocket in HCoV-NL63 is covered by a tight loop of residues 45–51 (<xref rid="Fig4" ref-type="fig">Fig. 4</xref>). Interestingly, the same region in SARS-CoV and HCoV-HKU1 adopts a secondary structure of 3<sub>10</sub> helix to maintain S2 pocket<sup><xref ref-type="bibr" rid="CR53">53</xref></sup>. Such difference might partially account for the increased variation of natural P2 residues as observed in the genomes of betacoronaviruses.<fig id="Fig4"><label>Figure 4</label><caption><p>S1 and S2 binding sites in HCoV-NL63 M<sup>pro</sup>.</p><p>The main chains of four human CoV M<sup>pro</sup> structures (HCoV-NL63: slate, HCoV-229E: cyan, SARS-CoV: magenta and HCoV-HKU1: yellow) are superimposed and displayed in the neightborhood of the substrate-binding site. The S1, S2 and S4 binding sites are labeled. The backbones are represented in worm form and inhibitor is shown in stick format of green color. Residues 45–51 are marked with a red oval (in dash line).</p></caption><graphic xlink:href="41598_2016_Article_BFsrep22677_Fig4_HTML" id="d29e1686"></graphic></fig></p></sec><sec id="Sec7"><title>P3, P4 and P5 positions</title><p>The P3 side chains of N3 inhibitor are both solvent exposed in the two protomers of HCoV-NL63 M<sup>pro</sup> (<xref rid="Fig2" ref-type="fig">Figs 2</xref>b and <xref rid="Fig3" ref-type="fig">3</xref>b). This is consistent with the fact that no specificity for any particular side chains exists at the P3 position of cleavage sites among CoV M<sup>pro</sup>s<sup><xref ref-type="bibr" rid="CR55">55</xref></sup>. Further, the NH and carbonyl oxygen of P3 backbone form two hydrogen bonds with the backbone carbonyl oxygen and NH of Glu166 respectively, which help anchor the N3 inhibitor to the HCoV-NL63 M<sup>pro</sup> at P3 location.</p><p>The P4 position of the inhibitor N3 is alanine and its side chain readily inserts into the relatively shallow P4 pocket (<xref rid="Fig3" ref-type="fig">Fig. 3b</xref>), forming hydrophobic interactions with Pro189. Also the backbone NH of alanine residue on N3 donates a 2.9-Å hydrogen bond to the carbonyl oxygen of Ser190 (<xref rid="Fig2" ref-type="fig">Fig. 2c</xref>). The isoxazole at P5 makes Van der Waals interactions with Gly168 and the backbone of residues Leu191 (<xref rid="Fig2" ref-type="fig">Fig. 2b</xref>). Overall, the pattern of interactions between N3 and HCoV-NL63 M<sup>pro</sup> at P3, P4 and P5 positions is similar to that between N3 and SARS-CoV M<sup>pro </sup><sup><xref ref-type="bibr" rid="CR49">49</xref></sup>.</p></sec><sec id="Sec8"><title>Structural conservation of M<sup>pro</sup> among clinical HCoV-NL63 isolates</title><p>So far, whole genomic sequences of as many as 30 strains of HCoV-NL63 have been deposited into NCBI database<sup><xref ref-type="bibr" rid="CR16">16</xref>,<xref ref-type="bibr" rid="CR44">44</xref>,<xref ref-type="bibr" rid="CR45">45</xref>,<xref ref-type="bibr" rid="CR60">60</xref>,<xref ref-type="bibr" rid="CR61">61</xref></sup>. These strains were isolated from patient specimens dated back to the past three decades from several countries, including The Netherlands, United States and China. High percentage of sequence variation among these clinically isolated HCoV-NL63 strains and evidence of <italic>in vivo</italic> recombination during co-infections with other CoVs have been documented, especially in the N-terminal domain of spike protein and nsp2/nsp3 region<sup><xref ref-type="bibr" rid="CR44">44</xref>,<xref ref-type="bibr" rid="CR60">60</xref></sup>. In order to determine the efficacy of inhibitor N3 against sequence variation accumulated during the circulation of HCoV-NL63 in human population, we assessed the level of sequence and structural conservation in the molecular target of this inhibitor among different clinical isolates. Sequence for M<sup>pro</sup> (nsp5) was retrieved from these HCoV-NL63 strains with available genomic sequences (<xref rid="MOESM1" ref-type="media">Supplementary Table S1</xref>) and alignment was performed to determine sequence variations among these clinical isolates. M<sup>pro</sup> sequence from strain Amsterdam I (NC_005831) was used as reference<sup><xref ref-type="bibr" rid="CR16">16</xref>,<xref ref-type="bibr" rid="CR44">44</xref></sup>. Overall, M<sup>pro</sup> is extremely conserved among clinical HCoV-NL63 strains isolated worldwide (<xref rid="MOESM1" ref-type="media">Supplementary Fig. S1</xref>), evidencing the enzyme’s essential role in viral replication. Sequence variation is only observed in 3 residues: His69 is mutated to Tyr in 13 strains isolated from United States, Cys221 is mutated to Arg among all the strains that have not been cultured <italic>in vitro</italic> and Met235 is mutated to Ile in only one strain isolated in United States (<xref rid="Fig5" ref-type="fig">Fig. 5a</xref>). When plotted onto the structure of HCoV-NL63 M<sup>pro</sup>, these 3 residues are all located in flexible regions, such as loop and molecular surface (<xref rid="Fig5" ref-type="fig">Fig. 5b</xref>). None of the 3 residues are directly interacting with inhibitor N3, which provides clear structural evidence to support the effectiveness of N3 against all the clinical strains of HCoV-NL63, in spite of significant genomic sequence variation and potential for recombination in viral transmission.<fig id="Fig5"><label>Figure 5</label><caption><p>Structural conservation of M<sup>pro</sup> among clinical HCoV-NL63 isolates.</p><p>(<bold>a</bold>) Summary of the 3 residues of variation among a total of 30 HCoV-NL63 strains. Amsterdam I is used as reference strain. Number of strains carrying one particular residue is labeled in bracket. (<bold>b</bold>) Three-dimensional representation of the 3 nonconserved residues from (<bold>a</bold>) mapped onto HCoV-NL63 M<sup>pro</sup> complex structure with inhibitor N3. Conserved residues are colored light orange, the 3 residues (His69, Cys221 and Met235) are colored cyan and inhibitor N3 is colored green.</p></caption><graphic xlink:href="41598_2016_Article_BFsrep22677_Fig5_HTML" id="d29e1827"></graphic></fig></p></sec><sec id="Sec9"><title>Inhibitor N3 targets a conserved site among human CoVs and their related zoonotic counterparts</title><p>The substrate-binding site of M<sup>pro</sup> has been shown as a conserved drug target for designing wide spectrum anti-CoV inhibitors<sup><xref ref-type="bibr" rid="CR48">48</xref>,<xref ref-type="bibr" rid="CR49">49</xref>,<xref ref-type="bibr" rid="CR51">51</xref>,<xref ref-type="bibr" rid="CR53">53</xref></sup>. Given the evidence of repetitive global epidemics (such as SARS and MERS) caused by zoonotic transmission<sup><xref ref-type="bibr" rid="CR11">11</xref>,<xref ref-type="bibr" rid="CR19">19</xref>,<xref ref-type="bibr" rid="CR20">20</xref>,<xref ref-type="bibr" rid="CR21">21</xref>,<xref ref-type="bibr" rid="CR22">22</xref>,<xref ref-type="bibr" rid="CR23">23</xref>,<xref ref-type="bibr" rid="CR24">24</xref></sup>, it is imperative to examine whether the substrate-binding site and the inhibition mechanism employed by inhibitor N3 are conserved between known human CoVs and their related zoonotic CoVs. We chose three most representative pairs of human CoVs and their zoonotic counterparts: HCoV-229E (NC_002645) and bovine coronavirus (BCoV, NC_003045), SARS-CoV (NC_004718) and SARS-related CoV isolated from bat (BtSARSr-CoV, KC881006), MERS-CoV (JX869059) and dromedary camel MERS-CoV (DcMERS-CoV, KJ713296). We then retrieved their M<sup>pro</sup> sequences from NCBI database. The direct bat ancestor of HCoV-NL63 has not been identified<sup><xref ref-type="bibr" rid="CR47">47</xref></sup>, therefore we only use HCoV-NL63 M<sup>pro</sup> sequence and its secondary structure presented in this study as reference. Sequence alignment, shown in <xref rid="Fig6" ref-type="fig">Fig. 6a</xref>, demonstrates significant homology in primary amino acid sequence among these 7 CoVs.<fig id="Fig6"><label>Figure 6</label><caption><p>Inhibitor N3 targets a conserved site among human and related zoonotic CoVs.</p><p>(<bold>a</bold>) Sequence alignment of three pairs of human CoVs and their related zoonotic counterparts: HCoV-229E and bovine coronavirus (BCoV), SARS-CoV and SARS-related coronavirus isolated from bat (BtSARSr-CoV), MERS-CoV and dromedary camel MERS-CoV (DcMERS-CoV). HCoV-NL63 and its secondary structure are used as reference for the alignment. Sequence alignment was performed using ClustalW2<sup><xref ref-type="bibr" rid="CR72">72</xref></sup> and figure was generated using ESPript 3.0<sup><xref ref-type="bibr" rid="CR73">73</xref></sup>. (<bold>b</bold>) Surface representation of conserved substrate-binding pockets from 7 CoV M<sup>pro</sup>s listed in (a). Background is HCoV-NL63 M<sup>pro</sup>. Red: identical residues among all seven CoV M<sup>pro</sup>; magenta: substitution in one CoV M<sup>pro</sup>; orange: substitution in two CoV M<sup>pro</sup>s. The residues forming the substrate-binding pocket are labeled. S1’: Leu27, Gly142; S1: Phe139, His163, Glu166, His172; S2: His41 Tyr53, Asp187; S4: Leu167, Gln192.</p></caption><graphic xlink:href="41598_2016_Article_BFsrep22677_Fig6_HTML" id="d29e1926"></graphic></fig></p><p>A closer examination at the substrate-binding pocket further reveals a conservative 3D architecture at this drug target (<xref rid="Fig6" ref-type="fig">Fig. 6b</xref>). Those residues, which are critical for pocket formation, such as Leu27, Gly142 for pocket S1’; Phe139, His163, Glu166, His172 for pocket S1; His41, Tyr53, Asp187 for pocket S2; Leu167, Gln192 for pocket S4, are strictly conserved among various CoVs (<xref rid="Fig6" ref-type="fig">Fig. 6a,b</xref>). Peptidomimetic inhibitor N3, through its Michael acceptor and well-designed side chains, snugly fits into the conserved substrate-binding pocket. The binding of N3 establishes a concerted interaction network, including a 1.8-Å covalent bond between Cβ of vinyl group on inhibitor N3 and the Sγ atom of Cys144, 7 hydrogen bonds and extensive hydrophobic interactions between N3 and the above residues critical for interplay. These findings demonstrate that inhibitor N3 could exert inhibitory effect towards a conserved site in CoVs both before and after zoonotic transmission. Therefore, in addition to its role in inhibiting known circulating human CoV species, inhibitor N3 might also serve as a lead compound for preclinical and clinical testing against potential future epidemic caused by CoV emerging from zoonotic origin.</p></sec></sec><sec id="Sec10"><title>Discussion</title><p>The world has experienced two global outbreaks of CoV infections since entering the 21<sup>st</sup> century<sup><xref ref-type="bibr" rid="CR5">5</xref>,<xref ref-type="bibr" rid="CR6">6</xref>,<xref ref-type="bibr" rid="CR7">7</xref>,<xref ref-type="bibr" rid="CR8">8</xref>,<xref ref-type="bibr" rid="CR9">9</xref>,<xref ref-type="bibr" rid="CR11">11</xref>,<xref ref-type="bibr" rid="CR12">12</xref>,<xref ref-type="bibr" rid="CR13">13</xref></sup>. Both SARS-CoV and MERS-CoV cause severe respiratory syndrome with high mortality rate<sup><xref ref-type="bibr" rid="CR10">10</xref>,<xref ref-type="bibr" rid="CR14">14</xref>,<xref ref-type="bibr" rid="CR15">15</xref></sup>. In addition, four more human CoVs, namely HCoV-NL63, HCoV-229E, HCoV-HKU1 and HCoV-OC43, have been identified as pathological agents for common cold<sup><xref ref-type="bibr" rid="CR9">9</xref>,<xref ref-type="bibr" rid="CR16">16</xref>,<xref ref-type="bibr" rid="CR17">17</xref></sup>. The lack of effective therapeutic and preventive strategies against human CoVs calls for immediate action of the scientific community<sup><xref ref-type="bibr" rid="CR9">9</xref>,<xref ref-type="bibr" rid="CR50">50</xref></sup>. We previously demonstrated that designing wide spectrum inhibitor at a conservative target is a viable method to develop anti-CoV therapeutics, given the high mutation and recombination rates observed in viral replication<sup><xref ref-type="bibr" rid="CR48">48</xref>,<xref ref-type="bibr" rid="CR49">49</xref>,<xref ref-type="bibr" rid="CR51">51</xref>,<xref ref-type="bibr" rid="CR53">53</xref></sup>. The ability of CoV to cross animal-human boundary provides further support to our strategy. Indeed, several human CoVs, including HCoV-NL63, SARS-CoV and MERS-CoV, have been linked to zoonotic CoVs which naturally infect hosts such as bats or dromedary camels<sup><xref ref-type="bibr" rid="CR20">20</xref>,<xref ref-type="bibr" rid="CR21">21</xref>,<xref ref-type="bibr" rid="CR22">22</xref>,<xref ref-type="bibr" rid="CR23">23</xref>,<xref ref-type="bibr" rid="CR24">24</xref>,<xref ref-type="bibr" rid="CR25">25</xref>,<xref ref-type="bibr" rid="CR46">46</xref></sup>. In current study, we use the M<sup>pro</sup> from HCoV-NL63 as our model molecule and present its crystal structure in complex with an inhibitor. Based on the structural detail at 2.85 Å resolution, the substrate-binding pocket of HCoV-NL63 M<sup>pro</sup> is conserved among three pairs of human and zoonotic CoVs (<xref rid="Fig6" ref-type="fig">Fig. 6</xref>). Several key residues at the subsites for substrate binding are completely identical among these seven coronaviruses, for example Phe139, His163, Glu166, His172 of S1; His41, Tyr53, Asp187 of S2; and Leu167, Gln192 of S4. These findings are consistent with the role of M<sup>pro</sup> in viral replication, which is essential to the proteolytic processing and maturation of replicase polyprotein (encoded by ORF1). Analysis of substrate recognition sequence, based on available whole genome sequences of all six human CoVs, provides additional evidence to the conservation of M<sup>pro</sup> substrate-binding pocket: the P1 position requires almost exclusively glutamine and the P2 position exhibits strong preference for hydrophobic residues such as Leu and Val (<xref rid="Tab3" ref-type="table">Table 3</xref>). Taken together, through designing of wide spectrum inhibitors at a conservative site on M<sup>pro</sup>, our study outlines a novel therapeutic approach of containing diseases caused by both existing and possible future emerging human CoVs.</p><p>A close examination of the interaction between HCoV-NL63 M<sup>pro</sup> and inhibitor N3 reveals structural details of inhibition mechanism. N3 is a synthetic peptidomimetic compound with Michael acceptor (<xref rid="Fig2" ref-type="fig">Fig. 2a</xref>), which achieves mechanism-based enzyme inactivation through forming an irreversible covalent bond with Cys144 of catalytic dyad (<xref rid="Fig2" ref-type="fig">Fig. 2b,c</xref>). The success of two serine protease inhibitors, telaprevir and boceprevir, in the treatment of hepatitis C virus (HCV) has underscored the importance of covalent inhibitors for targeting viral proteases<sup><xref ref-type="bibr" rid="CR62">62</xref></sup>. Both telaprevir and boceprevir are peptidomimetic inhibitors carry a warhead of α-ketoamide, which forms a covalent yet reversible bond with catalytic triad serine residue of HCV NS3∙4 A protease<sup><xref ref-type="bibr" rid="CR62">62</xref>,<xref ref-type="bibr" rid="CR63">63</xref></sup>. Michael acceptor has also been used as a warhead in pharmaceutical targeting against viral protease, for example rupintrivir was developed as an inhibitor for 3C protease of human rhinovirus and enterovirus<sup><xref ref-type="bibr" rid="CR50">50</xref>,<xref ref-type="bibr" rid="CR64">64</xref>,<xref ref-type="bibr" rid="CR65">65</xref></sup>. The backbone of N3 forms 7 hydrogen bonds with residues in the substrate-binding pocket (<xref rid="Fig2" ref-type="fig">Fig. 2c</xref>). The pockets accommodating N3 undergo gate-regulated switch to facilitate the binding of inhibitor N3 (<xref rid="Fig3" ref-type="fig">Fig. 3</xref>), an interesting phenomena initially observed in case of M<sup>pro</sup> from SARS-CoV<sup><xref ref-type="bibr" rid="CR49">49</xref></sup>.</p><p>In addition, the structure of HCoV-NL63 M<sup>pro</sup> exhibits several unique but interesting features. Examination of its natural cleavage sites based on HCoV-NL63 genomic sequence reveals an unusual histidine P1 residue between nsp13 and nsp14, which is unique to HCoV-NL63 and HCoV-HKU1 among all human CoVs<sup><xref ref-type="bibr" rid="CR37">37</xref></sup>. Such P1 anomaly is partially accommodated by the relatively smaller S1 pocket, as the size of its S1 pocket is comparable to that of HCoV-HKU1, but smaller than those of HCoV-229E and SARS-CoV. The P2 position seems to present genus specificity among alphacoronaviruses, as the P2 residues among all 11 natural cleavage sites are completely identical between HCoV-NL63 and HCoV-229E with a strong dominance of leucine. While large variation on P2 residue is observed in the genomes of human betacoronaviruses.</p><p>In summary, the crystal structure of HCoV-NL63 M<sup>pro</sup> complexed with inhibitor N3 has provided critical insight into the design of irreversible inhibitor carrying a Michael acceptor warhead. Through detailed sequence and structural comparison, this compound demonstrates feasibility as potential broad spectrum therapeutic agent for both existing and possibly emerging human CoVs. Further pharmaceutical development of such covalent peptidomimetic inhibitors would yield success in clinical management of human coronavirus diseases and public health preparedness for possible future pandemic.</p></sec><sec id="Sec11"><title>Methods</title><sec id="Sec12"><title>Protein expression and purification</title><p>Protein expression and purification of HCoV-NL63 main protease has been described previously<sup><xref ref-type="bibr" rid="CR66">66</xref></sup>. The coding sequence was subcloned into pGEX-6P-1 vector and transformed into BL21 (DE3) <italic>E. coli</italic> cells. Cell culture was first grown in LB medium containing 100 μg ml<sup>−1</sup> ampicillin at 37 °C until optical density (OD600) reached 0.6. Protein expression was then induced by adding isopropyl β-D-1-thiogalactopyranoside to a final concentration of 0.5 mM and further cultured at 16 °C for 16 hours. Cell pellets were harvested by centrifugation and resuspended in phosphate-buffered saline solution supplemented with 1 mM dithiothreitol (DTT) and 10% glycerol. Cell lysate was prepared using sonication and centrifugation (12,000 g, 50 min, 4 °C). GST-tagged HCoV-NL63 M<sup>pro</sup> fusion protein was bound to glutathione sepharose 4B affinity resin and GST tag was removed through on-column cleavage using commercial PreScission protease (GE Healthcare) at 4 °C for 18 hours. Recombinant HCoV-NL63 M<sup>pro</sup> protein was subject to an additional step of anion-exchange chromatography using HiTrap Q column (GE Healthcare) and was eluted with a linear gradient of 25 to 250 mM NaCl (20 mM Tris-HCl pH = 8.0, 10% glycerol, 1 mM DTT).</p></sec><sec id="Sec13"><title>Crystallization and data collection</title><p>Purified protein was supplemented with 10% DMSO and concentrated to 1 mg ml<sup>−1</sup>. Crystals of HCoV-NL63 M<sup>pro</sup> in complex with inhibitor N3 were produced by cocrystallization. Inhibitor N3 was added to HCoV M<sup>pro</sup> protein at a molar ratio between 3:1 and 5:1 and incubated at 4 °C for 4 hours. The complex was then centrifuged at 12,000 g for 10 min and concentrated to 10 mg ml<sup>−1</sup> in a buffer containing 10 mM HEPES pH 7.5, 150 mM NaCl, 1 mM DTT. Using hanging-drop vapor diffusion method at 16 °C, best crystals were obtained after 2 days using a reservoir solution containing 0.1 M HEPES pH 5.5, 10%(w/v) polyethylene glycol 8000, 4%(v/v) ethylene glycol (PDB entry 3TLO) and 0.1 M sodium citrate tribasic dehydrate pH 5.6, 1.0 M ammonium phosphate monobasic in a ratio of 80:20<sup><xref ref-type="bibr" rid="CR67">67</xref></sup>.</p><p>Data for HCoV-NL63 M<sup>pro</sup>-N3 complex was collected to a 2.85-Å resolution at 100 K on beamline 1W2B of Beijing Synchrotron Radiation Facility (BSRF), using a MAR165 charge-coupled device detector. The cryoprotectant solution contained 20% (v/v) glycerol, 10% (w/v) polyethylene glycol 8000, 4% (v/v) ethylene glycol and 0.1 M HEPES pH 5.5. All data integration and scaling were performed using HKL2000<sup><xref ref-type="bibr" rid="CR68">68</xref></sup>. The Matthews coefficient of the crystal suggested two molecules per asymmetric unit and the solvent content was 59.8%.</p></sec><sec id="Sec14"><title>Structure determination and analysis</title><p>The structure of HCoV-NL63 M<sup>pro</sup>-N3 complex was determined using molecular replacement from that of apo-form HCoV-NL63 M<sup>pro</sup> (PDB ID: 3TLO; C. P. Chuck & K. B. Wong, unpublished work). All cross-rotation and translation searches for molecular replacement were performed with Phaser<sup><xref ref-type="bibr" rid="CR69">69</xref></sup>. Cycles of manual adjustment using Coot<sup><xref ref-type="bibr" rid="CR70">70</xref></sup> and subsequent refinement using PHENIX<sup><xref ref-type="bibr" rid="CR71">71</xref></sup> led to a final model with a crystallographic R factor (R<sub>cryst</sub>) of 19.4% and a free R factor (R<sub>free</sub>) of 24.1% at 2.85-Å resolution.</p></sec><sec id="Sec15"><title>Enzymatic activity and inhibition assays</title><p>Enzymatic assay was performed using a fluorogenic substrate with consensus sequence of CoV M<sup>pro</sup>, MCA-AVLQSGFR-Lys(Dnp)-Lys-NH2 (>95% purity, GL Biochem Shanghai Ltd., Shanghai, China), as previously reported<sup><xref ref-type="bibr" rid="CR48">48</xref>,<xref ref-type="bibr" rid="CR49">49</xref>,<xref ref-type="bibr" rid="CR53">53</xref></sup>. Fluorescence intensity was monitored using a Fluoroskan Ascent instrument (Thermo Scientific, USA) with excitation and emission wavelengths of 320 nm and 405 nm, respectively. The assay was performed in a buffer solution consisted of 50 mM Tris-HCl (pH 7.3) and 1 mM EDTA at 30 °C. Kinetic parameters, including <italic>K</italic><sub><italic>m</italic></sub> and <italic>k</italic><sub><italic>cat</italic></sub> of apo HCoV-NL63 M<sup>pro</sup> and <italic>K</italic><sub><italic>i</italic></sub> and <italic>k</italic><sub><italic>3</italic></sub> of inhibitor N3, were determined using methods described in detail in our previous work<sup><xref ref-type="bibr" rid="CR49">49</xref></sup>.</p></sec></sec><sec id="Sec16"><title>Additional Information</title><p><bold>How to cite this article</bold>: Wang, F. <italic>et al.</italic> Structure of Main Protease from Human Coronavirus NL63: Insights for Wide Spectrum Anti-Coronavirus Drug Design. <italic>Sci. Rep.</italic><bold>6</bold>, 22677; doi: 10.1038/srep22677 (2016).</p></sec><sec sec-type="supplementary-material"><title>Electronic supplementary material</title><sec id="Sec17"><p><supplementary-material content-type="local-data" id="MOESM1"><media xlink:href="41598_2016_BFsrep22677_MOESM1_ESM.pdf"><caption><p>Table S1, Figure S1</p></caption></media></supplementary-material></p></sec></sec></body><back><fn-group><fn><p>Wang Fenghua and Chen Cheng contributed equally to this work.</p></fn></fn-group><ack><title>Acknowledgements</title><p>We thank Zengqiang Gao and Tianyi Zhang at beamline 1W2B of Beijing Synchrotron Radiation Facility (BSRF) for their technical support on data collection. This work was supported by National Key Basic Research Program of China (973 program) (No. 2015CB859800), National Natural Science Foundation of China (No. 31300150), Tianjin Marine Science and Technology Program (No. KJXH2014-16), Megapoject on Infectious Disease Control supported by the Ministry of Health of China (2013ZX10004601).</p></ack><notes notes-type="author-contribution"><title>Author Contributions</title><p>F.W. and C.C. performed the experiments. F.W., C.C., W.T., K.Y. and H.Y. analyzed the results. K.Y. and H.Y. wrote the manuscript with contributions from the other authors. 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