Structural and functional characterization of MERS coronavirus papain-like protease
Identifieur interne : 000387 ( Pmc/Corpus ); précédent : 000386; suivant : 000388Structural and functional characterization of MERS coronavirus papain-like protease
Auteurs : Min-Han Lin ; Shang-Ju Chuang ; Chiao-Che Chen ; Shu-Chun Cheng ; Kai-Wen Cheng ; Chao-Hsiung Lin ; Chiao-Yin Sun ; Chi-Yuan ChouSource :
- Journal of Biomedical Science [ 1021-7770 ] ; 2014.
Abstract
A new highly pathogenic human coronavirus (CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), has emerged in Jeddah and Saudi Arabia and quickly spread to some European countries since September 2012. Until 15 May 2014, it has infected at least 572 people with a fatality rate of about 30% globally. Studies to understand the virus and to develop antiviral drugs or therapy are necessary and urgent. In the present study, MERS-CoV papain-like protease (PLpro) is expressed, and its structural and functional consequences are elucidated.
Circular dichroism and Tyr/Trp fluorescence analyses indicated that the secondary and tertiary structure of MERS-CoV PLpro is well organized and folded. Analytical ultracentrifugation analyses demonstrated that MERS-CoV PLpro is a monomer in solution. The steady-state kinetic and deubiquitination activity assays indicated that MERS-CoV PLpro exhibits potent deubiquitination activity but lower proteolytic activity, compared with SARS-CoV PLpro. A natural mutation, Leu105, is the major reason for this difference.
Overall, MERS-CoV PLpro bound by an endogenous metal ion shows a folded structure and potent proteolytic and deubiquitination activity. These findings provide important insights into the structural and functional properties of coronaviral PLpro family, which is applicable to develop strategies inhibiting PLpro against highly pathogenic coronaviruses.
The online version of this article (doi:10.1186/1423-0127-21-54) contains supplementary material, which is available to authorized users.
Url:
DOI: 10.1186/1423-0127-21-54
PubMed: 24898546
PubMed Central: 4051379
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PMC:4051379Le document en format XML
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institution-id-type="ISNI">0000000104255914</institution-id><institution>Department of Life Sciences and Institute of Genome Sciences,</institution><institution>National Yang-Ming University,</institution></institution-wrap>Taipei, 112 Taiwan</nlm:aff></affiliation></author><author><name sortKey="Chen, Chiao Che" sort="Chen, Chiao Che" uniqKey="Chen C" first="Chiao-Che" last="Chen">Chiao-Che Chen</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.260770.4</institution-id><institution-id institution-id-type="ISNI">0000000104255914</institution-id><institution>Department of Life Sciences and Institute of Genome Sciences,</institution><institution>National Yang-Ming University,</institution></institution-wrap>Taipei, 112 Taiwan</nlm:aff></affiliation></author><author><name sortKey="Cheng, Shu Chun" sort="Cheng, Shu Chun" uniqKey="Cheng S" first="Shu-Chun" last="Cheng">Shu-Chun Cheng</name><affiliation><nlm:aff 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sortKey="Chou, Chi Yuan" sort="Chou, Chi Yuan" uniqKey="Chou C" first="Chi-Yuan" last="Chou">Chi-Yuan Chou</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.260770.4</institution-id><institution-id institution-id-type="ISNI">0000000104255914</institution-id><institution>Department of Life Sciences and Institute of Genome Sciences,</institution><institution>National Yang-Ming University,</institution></institution-wrap>Taipei, 112 Taiwan</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">24898546</idno><idno type="pmc">4051379</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4051379</idno><idno type="RBID">PMC:4051379</idno><idno type="doi">10.1186/1423-0127-21-54</idno><date when="2014">2014</date><idno type="wicri:Area/Pmc/Corpus">000387</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000387</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Structural and functional characterization of MERS coronavirus papain-like protease</title><author><name sortKey="Lin, Min Han" sort="Lin, Min Han" uniqKey="Lin M" first="Min-Han" last="Lin">Min-Han Lin</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.260770.4</institution-id><institution-id institution-id-type="ISNI">0000000104255914</institution-id><institution>Department of Life Sciences and Institute of Genome Sciences,</institution><institution>National Yang-Ming University,</institution></institution-wrap>Taipei, 112 Taiwan</nlm:aff></affiliation></author><author><name sortKey="Chuang, Shang Ju" sort="Chuang, Shang Ju" uniqKey="Chuang S" first="Shang-Ju" last="Chuang">Shang-Ju Chuang</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.260770.4</institution-id><institution-id institution-id-type="ISNI">0000000104255914</institution-id><institution>Department of Life Sciences and Institute of Genome Sciences,</institution><institution>National Yang-Ming University,</institution></institution-wrap>Taipei, 112 Taiwan</nlm:aff></affiliation></author><author><name sortKey="Chen, Chiao Che" sort="Chen, Chiao Che" uniqKey="Chen C" first="Chiao-Che" last="Chen">Chiao-Che Chen</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.260770.4</institution-id><institution-id institution-id-type="ISNI">0000000104255914</institution-id><institution>Department of Life Sciences and Institute of Genome Sciences,</institution><institution>National Yang-Ming University,</institution></institution-wrap>Taipei, 112 Taiwan</nlm:aff></affiliation></author><author><name sortKey="Cheng, Shu Chun" sort="Cheng, Shu Chun" uniqKey="Cheng S" first="Shu-Chun" last="Cheng">Shu-Chun Cheng</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.260770.4</institution-id><institution-id institution-id-type="ISNI">0000000104255914</institution-id><institution>Department of Life Sciences and Institute of Genome Sciences,</institution><institution>National Yang-Ming University,</institution></institution-wrap>Taipei, 112 Taiwan</nlm:aff></affiliation></author><author><name sortKey="Cheng, Kai Wen" sort="Cheng, Kai Wen" uniqKey="Cheng K" first="Kai-Wen" last="Cheng">Kai-Wen Cheng</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.260770.4</institution-id><institution-id institution-id-type="ISNI">0000000104255914</institution-id><institution>Department of Life Sciences and Institute of Genome Sciences,</institution><institution>National Yang-Ming University,</institution></institution-wrap>Taipei, 112 Taiwan</nlm:aff></affiliation></author><author><name sortKey="Lin, Chao Hsiung" sort="Lin, Chao Hsiung" uniqKey="Lin C" first="Chao-Hsiung" last="Lin">Chao-Hsiung Lin</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.260770.4</institution-id><institution-id institution-id-type="ISNI">0000000104255914</institution-id><institution>Department of Life Sciences and Institute of Genome Sciences,</institution><institution>National Yang-Ming University,</institution></institution-wrap>Taipei, 112 Taiwan</nlm:aff></affiliation></author><author><name sortKey="Sun, Chiao Yin" sort="Sun, Chiao Yin" uniqKey="Sun C" first="Chiao-Yin" last="Sun">Chiao-Yin Sun</name><affiliation><nlm:aff id="Aff2"><institution-wrap><institution-id institution-id-type="GRID">grid.454209.e</institution-id><institution-id institution-id-type="ISNI">0000000406392551</institution-id><institution>Department of Nephrology,</institution><institution>Chang-Gung Memorial Hospital,</institution></institution-wrap>Keelung, 204 Taiwan</nlm:aff></affiliation></author><author><name sortKey="Chou, Chi Yuan" sort="Chou, Chi Yuan" uniqKey="Chou C" first="Chi-Yuan" last="Chou">Chi-Yuan Chou</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.260770.4</institution-id><institution-id institution-id-type="ISNI">0000000104255914</institution-id><institution>Department of Life Sciences and Institute of Genome Sciences,</institution><institution>National Yang-Ming University,</institution></institution-wrap>Taipei, 112 Taiwan</nlm:aff></affiliation></author></analytic><series><title level="j">Journal of Biomedical Science</title><idno type="ISSN">1021-7770</idno><idno type="eISSN">1423-0127</idno><imprint><date when="2014">2014</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><sec><title>Backgrounds</title><p>A new highly pathogenic human coronavirus (CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), has emerged in Jeddah and Saudi Arabia and quickly spread to some European countries since September 2012. Until 15 May 2014, it has infected at least 572 people with a fatality rate of about 30% globally. Studies to understand the virus and to develop antiviral drugs or therapy are necessary and urgent. In the present study, MERS-CoV papain-like protease (PL<sup>pro</sup>) is expressed, and its structural and functional consequences are elucidated.</p></sec><sec><title>Results</title><p>Circular dichroism and Tyr/Trp fluorescence analyses indicated that the secondary and tertiary structure of MERS-CoV PL<sup>pro</sup> is well organized and folded. Analytical ultracentrifugation analyses demonstrated that MERS-CoV PL<sup>pro</sup> is a monomer in solution. The steady-state kinetic and deubiquitination activity assays indicated that MERS-CoV PL<sup>pro</sup> exhibits potent deubiquitination activity but lower proteolytic activity, compared with SARS-CoV PL<sup>pro</sup>. A natural mutation, Leu105, is the major reason for this difference.</p></sec><sec><title>Conclusions</title><p>Overall, MERS-CoV PL<sup>pro</sup> bound by an endogenous metal ion shows a folded structure and potent proteolytic and deubiquitination activity. These findings provide important insights into the structural and functional properties of coronaviral PL<sup>pro</sup> family, which is applicable to develop strategies inhibiting PL<sup>pro</sup> against highly pathogenic coronaviruses.</p></sec><sec><title>Electronic supplementary material</title><p>The online version of this article (doi:10.1186/1423-0127-21-54) contains supplementary material, which is available to authorized users.</p></sec></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Anderson, Lj" uniqKey="Anderson L">LJ Anderson</name></author><author><name sortKey="Baric, Rs" uniqKey="Baric R">RS Baric</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Chan, Jf" uniqKey="Chan J">JF Chan</name></author><author><name sortKey="Li, Ks" uniqKey="Li K">KS Li</name></author><author><name sortKey="To, Kk" uniqKey="To K">KK To</name></author><author><name sortKey="Cheng, Vc" uniqKey="Cheng V">VC 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<front><journal-meta><journal-id journal-id-type="nlm-ta">J Biomed Sci</journal-id><journal-id journal-id-type="iso-abbrev">J. Biomed. Sci</journal-id><journal-title-group><journal-title>Journal of Biomedical Science</journal-title></journal-title-group><issn pub-type="ppub">1021-7770</issn><issn pub-type="epub">1423-0127</issn><publisher><publisher-name>BioMed Central</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">24898546</article-id><article-id pub-id-type="pmc">4051379</article-id><article-id pub-id-type="publisher-id">588</article-id><article-id pub-id-type="doi">10.1186/1423-0127-21-54</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research</subject></subj-group></article-categories><title-group><article-title>Structural and functional characterization of MERS coronavirus papain-like protease</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Lin</surname><given-names>Min-Han</given-names></name><address><email>hantingpay@gmail.com</email></address><xref ref-type="aff" rid="Aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Chuang</surname><given-names>Shang-Ju</given-names></name><address><email>aspapsasp@hotmail.com</email></address><xref ref-type="aff" rid="Aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Chen</surname><given-names>Chiao-Che</given-names></name><address><email>march1990324@gmail.com</email></address><xref ref-type="aff" rid="Aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Cheng</surname><given-names>Shu-Chun</given-names></name><address><email>sama3665@hotmail.com</email></address><xref ref-type="aff" rid="Aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Cheng</surname><given-names>Kai-Wen</given-names></name><address><email>kevintzang@hotmail.com</email></address><xref ref-type="aff" rid="Aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Lin</surname><given-names>Chao-Hsiung</given-names></name><address><email>chlin2@ym.edu.tw</email></address><xref ref-type="aff" rid="Aff1">1</xref></contrib><contrib contrib-type="author" corresp="yes"><name><surname>Sun</surname><given-names>Chiao-Yin</given-names></name><address><email>fish3970@gmail.com</email></address><xref ref-type="aff" rid="Aff2">2</xref></contrib><contrib contrib-type="author" corresp="yes"><name><surname>Chou</surname><given-names>Chi-Yuan</given-names></name><address><email>cychou@ym.edu.tw</email></address><xref ref-type="aff" rid="Aff1">1</xref></contrib><aff id="Aff1"><label>1</label><institution-wrap><institution-id institution-id-type="GRID">grid.260770.4</institution-id><institution-id institution-id-type="ISNI">0000000104255914</institution-id><institution>Department of Life Sciences and Institute of Genome Sciences,</institution><institution>National Yang-Ming University,</institution></institution-wrap>Taipei, 112 Taiwan</aff><aff id="Aff2"><label>2</label><institution-wrap><institution-id institution-id-type="GRID">grid.454209.e</institution-id><institution-id institution-id-type="ISNI">0000000406392551</institution-id><institution>Department of Nephrology,</institution><institution>Chang-Gung Memorial Hospital,</institution></institution-wrap>Keelung, 204 Taiwan</aff></contrib-group><pub-date pub-type="epub"><day>4</day><month>6</month><year>2014</year></pub-date><pub-date pub-type="pmc-release"><day>4</day><month>6</month><year>2014</year></pub-date><pub-date pub-type="collection"><year>2014</year></pub-date><volume>21</volume><elocation-id>54</elocation-id><history><date date-type="received"><day>23</day><month>4</month><year>2014</year></date><date date-type="accepted"><day>19</day><month>5</month><year>2014</year></date></history><permissions><copyright-statement>© Lin et al.; licensee BioMed Central Ltd. 2014</copyright-statement><license><license-p>This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.</license-p></license></permissions><abstract id="Abs1"><sec><title>Backgrounds</title><p>A new highly pathogenic human coronavirus (CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), has emerged in Jeddah and Saudi Arabia and quickly spread to some European countries since September 2012. Until 15 May 2014, it has infected at least 572 people with a fatality rate of about 30% globally. Studies to understand the virus and to develop antiviral drugs or therapy are necessary and urgent. In the present study, MERS-CoV papain-like protease (PL<sup>pro</sup>) is expressed, and its structural and functional consequences are elucidated.</p></sec><sec><title>Results</title><p>Circular dichroism and Tyr/Trp fluorescence analyses indicated that the secondary and tertiary structure of MERS-CoV PL<sup>pro</sup> is well organized and folded. Analytical ultracentrifugation analyses demonstrated that MERS-CoV PL<sup>pro</sup> is a monomer in solution. The steady-state kinetic and deubiquitination activity assays indicated that MERS-CoV PL<sup>pro</sup> exhibits potent deubiquitination activity but lower proteolytic activity, compared with SARS-CoV PL<sup>pro</sup>. A natural mutation, Leu105, is the major reason for this difference.</p></sec><sec><title>Conclusions</title><p>Overall, MERS-CoV PL<sup>pro</sup> bound by an endogenous metal ion shows a folded structure and potent proteolytic and deubiquitination activity. These findings provide important insights into the structural and functional properties of coronaviral PL<sup>pro</sup> family, which is applicable to develop strategies inhibiting PL<sup>pro</sup> against highly pathogenic coronaviruses.</p></sec><sec><title>Electronic supplementary material</title><p>The online version of this article (doi:10.1186/1423-0127-21-54) contains supplementary material, which is available to authorized users.</p></sec></abstract><kwd-group xml:lang="en"><title>Keywords</title><kwd>MERS coronavirus</kwd><kwd>Papain-like protease</kwd><kwd>Deubiquitination</kwd><kwd>Antiviral target</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2014</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="Sec1"><title>Background</title><p>In September 2012, a new highly pathogenic human coronavirus (CoV)<sup>1</sup>, Middle East respiratory syndrome coronavirus (MERS-CoV), has emerged in Jeddah and Saudi Arabia and quickly spread to some European countries [<xref ref-type="bibr" rid="CR1">1</xref>–<xref ref-type="bibr" rid="CR3">3</xref>]. The virus causes symptoms similar to Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), yet involving an additional component of acute renal failure [<xref ref-type="bibr" rid="CR4">4</xref>]. Until 15 May 2014, it has infected at least 572 people with a fatality rate of about 30% globally (World Health Organization, global alert and response, <ext-link ext-link-type="uri" xlink:href="http://www.who.int/csr/don/2014_05_15_mers/en/">http://www.who.int/csr/don/2014_05_15_mers/en/</ext-link>). Recently, human-to-human transmission of MERS-CoV has been confirmed; albeit, a serological study of major livestock suggested dromedary camels also to be a possible host [<xref ref-type="bibr" rid="CR5">5</xref>, <xref ref-type="bibr" rid="CR6">6</xref>]. Nevertheless, these findings indicate that the virus have the opportunity to spread globally and pose a significant threat to world health and the economy. Therefore, studies to understand the virus and to develop antiviral drugs or therapy are necessary and urgent.</p><p>Like other CoVs, the MERS-CoV nonstructural polyproteins (pp1a and pp1ab) are cleaved by two types of viral cysteine proteases, a main protease (EC 3.4.22.69) and a papain-like protease (PL<sup>pro</sup>) (EC 3.4.22.46) [<xref ref-type="bibr" rid="CR7">7</xref>]. This processing is considered to be a suitable antiviral target because it is required for viral maturation. Unfortunately, initial screening of the existing SARS-CoV PL<sup>pro</sup> inhibitor, a benzodioxolane derivative against MERS-CoV PL<sup>pro</sup>, revealed no significant inhibition [<xref ref-type="bibr" rid="CR7">7</xref>]. The difference represents the requirement of further understanding the MERS-CoV PL<sup>pro</sup>. In addition to proteolytic activity, similar to those of SARS-CoV, NL63-CoV and murine hepatitis virus, MERS-CoV PL<sup>pro</sup> acts on both deubiquitination and ISG15-linked ISGylation [<xref ref-type="bibr" rid="CR8">8</xref>–<xref ref-type="bibr" rid="CR11">11</xref>]. As a viral deubiquitinating protease (DUB), MERS-CoV PL<sup>pro</sup> is able to deubiquitinate interferon regulatory factor 3 (IRF3), which can prevent its nuclear translocation and suppress production of interferon β [<xref ref-type="bibr" rid="CR10">10</xref>]. These studies support the multifunctional nature of coronaviral PL<sup>pro</sup>. Recently, with the crystal structure of SARS-CoV PL<sup>pro</sup> C112S mutant in complex with ubiquitin (Ub), we have demonstrated that Ub core (residue 1–72) makes mostly hydrophilic interactions with PL<sup>pro</sup>, while the Leu-Arg-Gly-Gly C-terminus of Ub is located in the catalytic cleft of PL<sup>pro</sup>, mimicking the P4-P1 residues [<xref ref-type="bibr" rid="CR12">12</xref>]. This bound pattern is similar to that of the ubiquitin-specific proteases (USPs), one of the five distinct DUB families [<xref ref-type="bibr" rid="CR13">13</xref>, <xref ref-type="bibr" rid="CR14">14</xref>].</p><p>The MERS-CoV PL<sup>pro</sup> domain in nsp3 of the pp1a proteins (residue 1484–1800) has been identified [<xref ref-type="bibr" rid="CR7">7</xref>, <xref ref-type="bibr" rid="CR10">10</xref>, <xref ref-type="bibr" rid="CR15">15</xref>]. Like other PL<sup>pro</sup>, there is a catalytic triad consisting of the residues Cys1592, His1759 and Asp1774. Homology modeling suggests that MERS-CoV PL<sup>pro</sup>, similar to other known PL<sup>pro</sup>, may have a right-hand-like architecture constituted by palm, thumb, and fingers domains, although their sequence identity are only about 30% [<xref ref-type="bibr" rid="CR12">12</xref>]. Furthermore, MERS-CoV PL<sup>pro</sup> is able to recognize and cleave at the LXGG consensus cleavage site, which is essential for most CoV PL<sup>pro</sup>-mediated processing [<xref ref-type="bibr" rid="CR10">10</xref>]. Despite this large body of knowledge on MERS-CoV PL<sup>pro</sup>, in the absence of detailed structural and functional characterization, the molecular basis for its catalytic mechanism remains poorly unknown.</p><p>Here, we expressed and purified the MERS-CoV PL<sup>pro</sup> by <italic>E. coli</italic> with high yield and high purity. The secondary, tertiary and quaternary structure of MERS-CoV PL<sup>pro</sup> was then investigated by circular dichroism (CD) spectroscopy, Tyr/Trp fluoresecence and analytical ultracentrifugation (AUC), respectively. The kinetic and DUB activity assays indicated that MERS-CoV PL<sup>pro</sup> exhibits potent DUB activity but lower proteolytic activity, compared with SARS-CoV PL<sup>pro</sup>. The present study provides a foundation for understanding the structural and biochemical properties of coronaviral PL<sup>pro</sup> family, which is applicable to develop strategies inhibiting PL<sup>pro</sup> for the effective control of highly pathogenic coronaviral infection.</p></sec><sec id="Sec2"><title>Methods</title><sec id="Sec3"><title>Expression plasmid construction</title><p>The sequence of MERS-CoV PL<sup>pro</sup> (GenBank accession number NC_019843.2; polyprotein residues 1484–1800) was synthesized (MDBio Inc.), digested by <italic>Nco</italic> I-<italic>Xho</italic> I and then inserted into the pET-28a(+) vector (Novagen). In the construct, the 6 x His tag was retained at the C-terminus. The reading frame was confirmed by sequencing.</p></sec><sec id="Sec4"><title>Expression and purification of MERS-CoV PL<sup>pro</sup></title><p>The expression vector was transformed into <italic>E. coli</italic> BL21 (DE3) cells (Novagen). For large scaled protein expression, cultures were grown in LB medium of 0.8 liter at 37°C for 4 h, induced with 0.4 mM isopropyl-β-<sub>D</sub>-thiogalactopyranoside, and incubated overnight at 20°C. After centrifuging at 6,000 x g at 4°C for 15 min, the cell pellets were resuspended in lysis buffer (20 mM Tris, pH 8.5, 250 mM NaCl, 5% glycerol, 0.2% Triton X-100, and 2 mM β-mercaptoethanol) and then lysed by sonication. The crude extract was then centrifuged at 12,000 x g at 4°C for 25 min to remove the insoluble pellet. The supernatant was incubated with 1-ml Ni-NTA beads at 4°C for 1 h and then loaded into an empty column. After allowing the supernatant to flow through, the beads were washed with washing buffer (20 mM Tris, pH 8.5, 250 mM NaCl, 8 mM imidazole, and 2 mM β-mercaptoethanol), and the protein was eluted with elution buffer (20 mM Tris, pH 8.5, 30 mM NaCl, 150 mM imidazole, and 2 mM β-mercaptoethanol). The protein was then loaded onto a S-100 gel-filtration column (GE Healthcare) equilibrated with running buffer (20 mM Tris, pH 8.5, 100 mM NaCl, and 2 mM dithiothreitol). The purity of the fractions collected was analyzed by SDS-PAGE and the protein was concentrated to 30 mg/ml by Amicon Ultra-4 10-kDa centrifugal filter (Millipore).</p></sec><sec id="Sec5"><title>Circular dichroism spectroscopy</title><p>CD spectra of the recombinant MERS-CoV PL<sup>pro</sup> using a JASCO J-810 spectropolarimeter showed measurements from 250 to 190 nm at 20°C in 50 mM phosphate pH 6.5. The protein concentration was 1.0 mg/ml. In wavelength scanning, the width of the cuvette was 0.1 mm. The far-UV CD spectrum data were analyzed with the CDSSTR program [<xref ref-type="bibr" rid="CR16">16</xref>, <xref ref-type="bibr" rid="CR17">17</xref>]. In this analysis, the α-helix, β-sheet, and random coil were split. To estimate the goodness-of-fit, the normalized root mean square deviation was calculated.</p></sec><sec id="Sec6"><title>Spectrofluorimetric analysis</title><p>The fluorescence spectra of the enzyme at 1 μM were monitored in a Perkin-Elmer LS50B luminescence spectrometer at 25°C. The excitation wavelength was set at 280 nm, and the fluorescence emission spectrum was scanned from 300 to 400 nm. Measurement in the maximal peak, intensity, and average emission wavelength were used to confirm the protein folding [<xref ref-type="bibr" rid="CR18">18</xref>, <xref ref-type="bibr" rid="CR19">19</xref>].</p></sec><sec id="Sec7"><title>Analytical ultracentrifugation analysis</title><p>The AUC experiments were performed on a XL-A analytical ultracentrifuge (Beckman Coulter) using an An-50 Ti rotor [<xref ref-type="bibr" rid="CR12">12</xref>, <xref ref-type="bibr" rid="CR19">19</xref>–<xref ref-type="bibr" rid="CR22">22</xref>]. The sedimentation velocity experiments were performed using a double-sector <italic>epon</italic> charcoal-filled centerpiece at 20°C with a rotor speed of 42,000 rpm. Protein solutions of MERS-CoV PL<sup>pro</sup> (1.0 mg/ml) (330 μl) and reference (370 μl) solutions were loaded into the centerpiece, respectively. The absorbance at 280 nm was monitored in a continuous mode with a time interval of 300 s and a step size of 0.003 cm. Multiple scans at different time intervals were then fitted to a continuous c(s) distribution model using the SEDFIT program [<xref ref-type="bibr" rid="CR23">23</xref>]. All size-and-shape distributions were analyzed at a confidence level of p = 0.95 by maximal entropy regularization and a resolution N of 200 with sedimentation coefficients between 0 and 20 S or molar mass between 0 and 1000 kDa.</p></sec><sec id="Sec8"><title>Steady-state kinetic analysis</title><p>The peptidyl substrate, Dabcyl–FRLKGGAPIKGV–Edans, was used to measure the enzymatic activity of MERS-CoV PL<sup>pro</sup> and its mutants throughout the course of the study as described [<xref ref-type="bibr" rid="CR24">24</xref>]. Specifically, the enhanced fluorescence emission upon substrate cleavage was monitored at excitation and emission wavelengths of 329 and 520 nm, respectively, in a PerkinElmer LS 50B luminescence spectrometer. Fluorescence intensity was converted to the amount of hydrolyzed substrate using a standard curve drawn from the fluorescence measurements of well-defined concentrations of Dabcyl–FRLKGG and APIKGV–Edans peptides in a 1:1 ratio. This will also correct for the inner filter effect of the substrate. For the kinetic analysis, the reaction mixture contained 4–50 μM peptide substrate in 50 mM phosphate pH 6.5 in a total volume of 1 mL. After the addition of the enzyme to the reaction mixture, the increase in fluorescence was continuously monitored at 30°C. The increase in fluorescence was linear for at least 3 min, and thus the slope of the line represented the initial velocity (<italic>v</italic>). The steady-state kinetic parameters of the enzyme were determined by fitting the Michaelis–Menten equation (eq. <xref rid="Equ1" ref-type="">1</xref>) to the initial velocity data<disp-formula id="Equ1"><label>1</label><graphic xlink:href="12929_2014_Article_588_Equ1_HTML.gif" position="anchor"></graphic></disp-formula></p><p>in which <italic>k</italic><sub>cat</sub> is the rate constant, [<italic>E</italic>] and [<italic>S</italic>] denote the enzyme and substrate concentration, and <italic>K</italic><sub>m</sub> is the Michaelis-Menten constant for the interaction between the peptide substrate and the enzyme.</p></sec><sec id="Sec9"><title>Deubiquitination assay</title><p>The fluorogenic substrate Ub-7-amino-4-trifluoro-methylcoumarin (Ub-AFC) (Boston Biochem) added at 0.5 or 1.0 μM to 50 mM phosphate pH 6.5 was used for deubiquitination assays as described [<xref ref-type="bibr" rid="CR12">12</xref>]. The enzymatic activity at 30°C was determined by continuously monitoring the fluorescence emission and excitation wavelength of 350 and 485 nm, respectively.</p></sec></sec><sec id="Sec10"><title>Results and discussion</title><sec id="Sec11"><title>Recombinant MERS-CoV PL<sup>pro</sup> preparation</title><p>To date, there are still no studies describing the expression and purification of MERS-CoV PL<sup>pro</sup> proteins. In the present study, the expression vector was constructed and then various <italic>E. coli</italic>. strains such as BL21 (DE3) STAR (Invitrogen) and Rosetta (DE3) (Novagen) were used to explore heterologous expression of MERS-CoV PL<sup>pro</sup>. Finally, it was found that the STAR strain showed the best expression efficiency. After expressing the protein in <italic>E. coli</italic> and purification by nickel affinity chromatography and gel-filtration, the purity of recombinant PL<sup>pro</sup> was about 99% (Figure <xref rid="Fig1" ref-type="fig">1</xref>A). The size of the recombinant MERS-CoV PL<sup>pro</sup> was found to be between 30 and 45 kDa, which conforms to the theoretical mass (36.5 kDa). The typical yield was about 42 mg after purification from 0.8 liter of <italic>E. coli</italic> culture (Table <xref rid="Tab1" ref-type="table">1</xref>). After gel-filtration chromatography, the specific proteolytic activity of PL<sup>pro</sup> was 4 U/mg, increased by 5-fold, with 49.4% recovery rate.<fig id="Fig1"><label>Figure 1</label><caption><p><bold>Expression and purification of recombinant MERS</bold>-<bold>CoV PL</bold><sup><bold>pro</bold></sup><bold>. (A)</bold> Protein identification by SDS-PAGE. M: molecular marker. Lane 1–4: cytoplasmic fraction, flow-through, elution from the nickel affinity column and protein fraction from S-100 gel-filtration column. <bold>(B)</bold> and <bold>(C)</bold> Protein sequence identification by mass spectrometry. The PL<sup>pro</sup> was digested by trypsin and then analyzed by MALDI mass spectrometry. There are 15 matched peptides observed <bold>(B)</bold> and 60% sequence coverage are shown in bold red <bold>(C)</bold>.</p></caption><graphic xlink:href="12929_2014_Article_588_Fig1_HTML" id="d29e749"></graphic></fig></p><table-wrap id="Tab1"><label>Table 1</label><caption><p><bold>Purification of MERS</bold>-<bold>CoV PL</bold><sup><bold>pro</bold></sup><bold>from</bold><bold><italic>E. coli</italic></bold></p></caption><table frame="hsides" rules="groups"><thead><tr><th>Step</th><th>Total protein (mg)</th><th>Total activity (U<sup>a</sup>)</th><th>Specific activity (U/mg protein)</th><th>Purification (-fold)</th><th>Recovery (%)</th></tr></thead><tbody><tr><td>Cytoplasmic fraction</td><td>426</td><td>340</td><td>0.80</td><td>1</td><td>100</td></tr><tr><td>Ni affinity chromatography</td><td>64.4</td><td>221</td><td>3.43</td><td>4.3</td><td>65</td></tr><tr><td>Gel-filtration by S-100 column</td><td>41.9</td><td>168</td><td>4.01</td><td>5.0</td><td>49.4</td></tr></tbody></table><table-wrap-foot><p><sup>a</sup>One unit is defined as the amount of enzyme required to catalyze the cleavage of 1 nmole of peptidyl substrate (Dabcyl-FRLKGGAPIKGV-Edans) per minute at 30°C.</p></table-wrap-foot></table-wrap><p>Furthermore, the recombinant MERS-CoV PL<sup>pro</sup> was digested by trypsin and then analyzed by MALDI mass spectrometry to confirm the amino acid sequence (Additional file <xref rid="MOESM1" ref-type="media">1</xref>: Figure S1). The molecular weight of fifteen peptides, which covered 60% amino acid sequence, was observed and confirmed (Figure <xref rid="Fig1" ref-type="fig">1</xref>B and Figure <xref rid="Fig1" ref-type="fig">1</xref>C). It indicated that our expression and purification of MERS-CoV PL<sup>pro</sup> by <italic>E. coli</italic> is successful. For convenience, in the present studies, the MERS-CoV PL<sup>pro</sup> domain (polyprotein 1a 1484–1800) is numbered to residue 2 to 317, while the first residue is a methionine.</p></sec><sec id="Sec12"><title>Secondary, tertiary and quaternary structure analysis of MERS-CoV PL<sup>pro</sup></title><p>Next, secondary, tertiary and quaternary structures of MERS-CoV PL<sup>pro</sup> were investigated, respectively. CD measurement displayed a spectrum which shows negative ellipticity between 240 and 205 nm and positive between 205 and 190 nm (Figure <xref rid="Fig2" ref-type="fig">2</xref>). After analyzed by CDSSTR method [<xref ref-type="bibr" rid="CR16">16</xref>], the best-fit result showed that MERS-CoV PL<sup>pro</sup> has 23% of α-helix, 31% of β-sheet, and 46% of random coil. The consist is close to that of SARS-CoV PL<sup>pro</sup> (pdb code: 4M0W) by X-ray crystallography, which has 26% of α-helix, 36% of β-sheet, and 38% of random coil [<xref ref-type="bibr" rid="CR12">12</xref>]. It suggests that both PL<sup>pro</sup> may have a similar scaffold.<fig id="Fig2"><label>Figure 2</label><caption><p><bold>CD spectrum of MERS</bold>-<bold>CoV PL</bold><sup><bold>pro</bold></sup><bold>.</bold> The protein of 1 mg/ml was suspended in 50 mM phosphate pH 6.5 and the CD values were measured from 240 to 190 nm at 20°C. The obtained spectrum is shown as close circles and the best fit by CDSSTR [<xref ref-type="bibr" rid="CR16">16</xref>] is shown by solid line. The normalized root mean square deviation is 0.015.</p></caption><graphic xlink:href="12929_2014_Article_588_Fig2_HTML" id="d29e936"></graphic></fig></p><p>The Tyr/Trp fluorescence of MERS-CoV PL<sup>pro</sup> at the phosphate buffer without or with 9 M urea were also identified (Figure <xref rid="Fig3" ref-type="fig">3</xref>). The measurement indicated that the fluorescent intensity of native PL<sup>pro</sup> (Figure <xref rid="Fig3" ref-type="fig">3</xref>, close circles) shows a 70% increase, as compared with that of the denatured form in urea (Figure <xref rid="Fig3" ref-type="fig">3</xref>, open circles). On the other hand, the fluorescence emission spectrum of the native MERS-CoV PL<sup>pro</sup> shows a maximum at 336 nm, while that of the unfolded one shifts to 340 nm. The tendency is similar to that of SARS-CoV PL<sup>pro</sup> [<xref ref-type="bibr" rid="CR19">19</xref>] and suggests a folded structure. Next, we also performed AUC experiments to characterize the quaternary structure of MERS-CoV PL<sup>pro</sup>. Figure <xref rid="Fig4" ref-type="fig">4</xref>A shows a typical absorbance trace at 280 nm of the PL<sup>pro</sup> during the experiment. After fitting the signals to a continuous size-distribution model, it was clear that the PL<sup>pro</sup> was monomeric with a sedimentation coefficient of 2.8 S and molar mass of 35.5 kDa (Figure <xref rid="Fig4" ref-type="fig">4</xref>B and Figure <xref rid="Fig4" ref-type="fig">4</xref>C), consistent with that for SARS-CoV PL<sup>pro</sup> [<xref ref-type="bibr" rid="CR12">12</xref>, <xref ref-type="bibr" rid="CR19">19</xref>]. All of these biophysical observation confirmed that the PL<sup>pro</sup> of MERS-CoV and SARS-CoV should have a very similar structure; albeit they only show 30% sequence identity and 50% similarity [<xref ref-type="bibr" rid="CR12">12</xref>]. Recent studies hypothesized that the homology model of MERS-CoV PL<sup>pro</sup>, like other coronaviral PL<sup>pro</sup>, is a right-hand-like architecture consisting of palm, thumb and fingers domains [<xref ref-type="bibr" rid="CR10">10</xref>, <xref ref-type="bibr" rid="CR25">25</xref>].<fig id="Fig3"><label>Figure 3</label><caption><p><bold>Fluorescence spectrum of MERS</bold>-<bold>CoV PL</bold><sup><bold>pro</bold></sup><bold>.</bold> The protein of 1 μM was dissolved in 50 mM phosphate pH 6.5 (closed circles) or 9 M urea (open circles) and excited with 280 nm UV light. The protein fluorescence emission was monitored from 300 to 400 nm at 25°C.</p></caption><graphic xlink:href="12929_2014_Article_588_Fig3_HTML" id="d29e1030"></graphic></fig><fig id="Fig4"><label>Figure 4</label><caption><p><bold>The continuous size distribution change of MERS-CoV PL</bold><sup><bold>pro</bold></sup><bold>. (A)</bold> Traces of absorbance at 280 nm of the enzyme in the 50 mM phosphate pH 6.5 during the SV experiment. The protein concentration was 1 mg/ml. For clarity, only every four scan is shown. The symbols represent experimental data and the lines are the results obtained after fitted to the Lamm equation using the SEDFIT program [<xref ref-type="bibr" rid="CR23">23</xref>, <xref ref-type="bibr" rid="CR26">26</xref>]. <bold>(B)</bold> and <bold>(C)</bold> show the continuous c(s) and c(M) distribution of PL<sup>pro</sup>, respectively. The residual bitmap of the raw data and the best-fit results are shown in the inset.</p></caption><graphic xlink:href="12929_2014_Article_588_Fig4_HTML" id="d29e1062"></graphic></fig></p></sec><sec id="Sec13"><title>Proteolytic activity of MERS-CoV PL<sup>pro</sup></title><p>Besides the structural similarity, previous studies have suggested that MERS-CoV PL<sup>pro</sup> is also a multifunctional enzyme with protease, deubiquitinating and interferon antagonist activities [<xref ref-type="bibr" rid="CR10">10</xref>]. MERS-CoV PL<sup>pro</sup> has a catalytic triad which is able to recognize and cleave at LXGG consensus cleavage sites; however, the detail enzyme kinetic mechanism is not known. Here we used the peptidyl substrate, Dabcyl–FRLKGGAPIKGV–Edans, to measure the proteolytic activity of MERS-CoV PL<sup>pro</sup> (Figure <xref rid="Fig5" ref-type="fig">5</xref>A and Table <xref rid="Tab2" ref-type="table">2</xref>). Interestingly, compared with that of SARS-CoV, MERS-CoV PL<sup>pro</sup> is less active, with a 22-fold loss in k<sub>cat</sub>/K<sub>m</sub>, as a result of a 27.5-fold loss in k<sub>cat</sub> and 1.3-fold loss in K<sub>m</sub>. According to the sequence alignment and homology modeling, most important residues for the catalysis, including the catalytic triad, Cys110-His277-Asp292 and the residues for substrate P4-P1 binding, Asp164, Pro249, and Gly276 (Asp165, Pro249, and Gly272 in SARS-CoV PL<sup>pro</sup>) are highly conserved (Figure <xref rid="Fig6" ref-type="fig">6</xref>) [<xref ref-type="bibr" rid="CR10">10</xref>, <xref ref-type="bibr" rid="CR12">12</xref>]. Previous studies have confirmed that Y265F mutant of SARS-CoV PL<sup>pro</sup> still maintained comparable proteolytic activity with the wild-type [<xref ref-type="bibr" rid="CR12">12</xref>]. It indicates that the equivalent residue in MERS-CoV PL<sup>pro</sup>, Phe268, is able to make a hydrophobic contact with the substrate P4 residue (Ub-Leu73). Furthermore, although different to the residue Tyr269 of SARS-CoV PL<sup>pro</sup>, the equivalent residue Glu272 of MERS-CoV PL<sup>pro</sup>, whose carboxyl group can point toward outside the hydrophobic pocket, may not interfere the binding of substrate P4 residue (Figure <xref rid="Fig6" ref-type="fig">6</xref>). By contrast, as a putative oxyanion bound residue (Tyr107 in SARS-CoV PL<sup>pro</sup>) [<xref ref-type="bibr" rid="CR15">15</xref>], the equivalent residue Leu105 of MERS-CoV PL<sup>pro</sup> cannot provide any hydrogen bonding interaction with oxyanion (Figure <xref rid="Fig6" ref-type="fig">6</xref>). It will disfavor the formation of tetrahedral intermediate. Otherwise, different to the Leu163 of SARS-CoV PL<sup>pro</sup>, the distinct circular structure of the equivalent residue Pro162 of MERS-CoV PL<sup>pro</sup> may be too short to hover above the active site for substrate binding and serve to enhance the nucleophilicity of the catalytic triad residue, Cys110 (Figure <xref rid="Fig6" ref-type="fig">6</xref>). These two point mutations in MERS-CoV PL<sup>pro</sup> may significantly lower the catalytic efficiency.<fig id="Fig5"><label>Figure 5</label><caption><p><bold>Proteolytic and DUB activity assay of MERS-CoV PL</bold><sup><bold>pro</bold></sup><bold>and its mutants.</bold> Panel <bold>(A)</bold> shows the plot of initial velocities versus the concentration of peptidyl substrate, Dabcyl-FRLKGGAPIKGV-Edans. The concentration of the wild-type MERS-CoV PL<sup>pro</sup> (by circles), the L105W (by triangles) and P162L mutants (by squares) was 1, 0.1 and 10 μM, respectively. The line represented the best-fit results according to the Michaelis-Menten equation (Eq. <xref rid="Equ1" ref-type="">1</xref>). The kinetic parameters derived are shown in Table <xref rid="Tab2" ref-type="table">2</xref>. <bold>(B)</bold> DUB activity analysis. The fluorogenic substrate Ub-AFC (1 μM) was used as the substrate. For comparison, both DUB activity of SARS-CoV and MERS-CoV PL<sup>pro</sup> was tested. The protein concentration was 0.17 μM. Besides, the inhibition of MERS-CoV PL<sup>pro</sup> by 10–50 mM EDTA or 50 μM Zn<sup>2+</sup> were also clarified.</p></caption><graphic xlink:href="12929_2014_Article_588_Fig5_HTML" id="d29e1202"></graphic></fig></p><table-wrap id="Tab2"><label>Table 2</label><caption><p><bold>The kinetic parameters and DUB activity of MERS</bold>-<bold>CoV PL</bold><sup><bold>pro</bold></sup></p></caption><table frame="hsides" rules="groups"><thead><tr><th rowspan="3">Proteins</th><th colspan="3">Peptide cleavage</th><th>Deubiquitination</th></tr><tr><th>K<sub>m</sub></th><th><bold><italic>k</italic></bold><sub>cat</sub></th><th><bold><italic>k</italic></bold><sub>cat</sub>/K<sub>m</sub></th><th rowspan="2">Activity (Intensity/s)<sup>b</sup></th></tr><tr><th>(μM)<sup>a</sup></th><th>(10<sup>-2</sup> s<sup>-1</sup>)<sup>a</sup></th><th>(10<sup>-3</sup> s<sup>-1</sup> μM<sup>-1</sup>)</th></tr></thead><tbody><tr><td>MERS-CoV PL<sup>pro</sup></td><td></td><td></td><td></td><td></td></tr><tr><td>Wild-type</td><td>19.2 ± 2.6</td><td>0.4 ± 0.02</td><td>0.2 ± 0.03</td><td>0.11 ± 0.02</td></tr><tr><td>L105W mutant</td><td>35.7 ± 3.8</td><td>16.5 ± 0.9</td><td>4.6 ± 0.6</td><td>0.11 ± 0.01</td></tr><tr><td>P162L mutant</td><td>30.8 ± 8.0</td><td>0.01 ± 0.001</td><td>0.003 ± 0.001</td><td>0.004 ± 0.001</td></tr><tr><td>SARS-CoV PL<sup>pro</sup></td><td>25.2 ± 5.1<sup>c</sup></td><td>11 ± 2<sup>c</sup></td><td>4.4 ± 1.2<sup>c</sup></td><td>0.12 ± 0.02</td></tr></tbody></table><table-wrap-foot><p><sup>a</sup>Kinetic data of MERS-CoV PL<sup>pro</sup> and its mutants were fitted to the Michaelis-Menten equation (Eq. <xref rid="Equ1" ref-type="">1</xref>). The R<sub>sqr</sub> were from 0.986 to 0.997, respectively. All the assays were repeated several times to ensure reproducibility.</p><p><sup>b</sup>Fixed concentrations of Ub-AFC (0.5 μM) and PL<sup>pro</sup> (0.17 μM) were used.</p><p><sup>c</sup>The values were from our previous studies [<xref ref-type="bibr" rid="CR24">24</xref>].</p></table-wrap-foot></table-wrap><fig id="Fig6"><label>Figure 6</label><caption><p><bold>Putative active site of MERS-CoV PL</bold><sup><bold>pro</bold></sup><bold>.</bold> The model structure of MERS-CoV PL<sup>pro</sup> (in cyan) was generated by SWISS-MODEL [<xref ref-type="bibr" rid="CR27">27</xref>] and then overlaid with the structure of SARS-CoV PL<sup>pro</sup> (in grey) in complex with Ub (in yellow) (PDB code: 4M0W). The residues are shown as sticks and hydrogen bonding and ion-pair interactions are indicated by red dashed lines. Four residues of SARS-CoV PL<sup>pro</sup>, Trp107, Leu163, Tyr265 and Tyr269, are labeled in black. The figure was produced using PyMol (<ext-link ext-link-type="uri" xlink:href="http://www.pymol.org">http://www.pymol.org</ext-link>).</p></caption><graphic xlink:href="12929_2014_Article_588_Fig6_HTML" id="d29e1436"></graphic></fig><p>To verify this, we produced the L105W and P162L mutants of MERS-CoV PL<sup>pro</sup>, and our kinetic data showed that the L105W mutant has a 23-fold increase in activity measured based on k<sub>cat</sub>/K<sub>m</sub>, as a result of a 41-fold increase in k<sub>cat</sub> and 1.9-fold increase in K<sub>m</sub> (Figure <xref rid="Fig5" ref-type="fig">5</xref>A and Table <xref rid="Tab2" ref-type="table">2</xref>). The results conform to our prediction. However, in contrast, the P162L mutant has a 67-fold loss in k<sub>cat</sub>/K<sub>m</sub>, as a result of a 40-fold loss in k<sub>cat</sub> and 1.6-fold increase in K<sub>m</sub> (Figure <xref rid="Fig5" ref-type="fig">5</xref>A and Table <xref rid="Tab2" ref-type="table">2</xref>). It suggests the requirement of the Proline residue in this site, although the reason is still not known. Nevertheless, the significant activity recovery by L105W mutation confirms the essential role of this residue on coronaviral PL<sup>pro</sup> catalysis. Theoretically, PL<sup>pro</sup> with lower proteolytic activity may result in late maturation of viral nsp1, nsp2, and nsp3 proteins; nonetheless, its influence on MERS-CoV remains unknown.</p></sec><sec id="Sec14"><title>DUB activity of MERS-CoV PL<sup>pro</sup></title><p>To characterize the DUB activity of MERS-CoV PL<sup>pro</sup>, the fluorogenic substrate Ub-AFC was used. Interestingly, in contrast with its rather low proteolytic activity, MERS-CoV PL<sup>pro</sup> shows comparable DUB activity to SARS-CoV PL<sup>pro</sup> (Table <xref rid="Tab2" ref-type="table">2</xref> and Figure <xref rid="Fig5" ref-type="fig">5</xref>B). It suggests that the two PL<sup>pro</sup> may show similar binding ability to the Ub core domain (residue 1–72). However, it is inconsistent with our previous observation on the structure of SARS-CoV PL<sup>pro</sup> in complex with Ub [<xref ref-type="bibr" rid="CR12">12</xref>]. As mimicking the equivalent residue of MERS-CoV PL<sup>pro</sup>, the arginine mutation of a key residue for Ub core domain binding, Glu168, can result in unstable binding of SARS-CoV PL<sup>pro</sup> and Ub and significant loss of DUB activity [<xref ref-type="bibr" rid="CR12">12</xref>]. To verify this inconsistency, a structure of MERS-CoV PL<sup>pro</sup> in complex with Ub is quite necessary.</p><p>Structural characterization of type 1 and type 2 PL<sup>pro</sup> have revealed that there are four cysteine residues coordinating to a zinc ion within the fingertips region in the finger domain [<xref ref-type="bibr" rid="CR25">25</xref>, <xref ref-type="bibr" rid="CR28">28</xref>]. Remove of zinc from SARS-CoV PL<sup>pro</sup> will cause the tertiary structure more unstable and lead to less active [<xref ref-type="bibr" rid="CR19">19</xref>]. Based on sequence alignment, MERS-CoV PL<sup>pro</sup> also has four cysteine residues (Cys190, Cys193, Cys225 and Cys227) on the corresponding position. Here the DUB activity of MERS-CoV PL<sup>pro</sup> in various EDTA was examined to delineate the possible metal ion effect. The activity was 79% in 10 mM, and 72% left in 50 mM EDTA (Figure <xref rid="Fig5" ref-type="fig">5</xref>B). These results suggest the existence of endogenous metal ion, which is beneficial for its DUB activity. By the way, it has been clarified that exogenous zinc ion can efficiently inhibit SARS-CoV PL<sup>pro</sup> with the IC<sub>50</sub> value of 1.3 μM [<xref ref-type="bibr" rid="CR24">24</xref>, <xref ref-type="bibr" rid="CR29">29</xref>]. Here we also confirmed the potent inhibitory effect of zinc ion on MERS-CoV PL<sup>pro</sup> (Figure <xref rid="Fig5" ref-type="fig">5</xref>B); whereas the mechanism of this inhibition by zinc is not yet understood.</p></sec></sec><sec id="Sec15"><title>Conclusions</title><p>In summary, following our protocol, active MERS-CoV PL<sup>pro</sup> can be expressed by <italic>E. coli</italic> and purified with high yield and high purity. The secondary, tertiary and quaternary structural studies concluded that MERS-CoV PL<sup>pro</sup> has a similar scaffold to other coronaviral PL<sup>pro</sup>, as a right-hand-like architecture consisting of palm, thumb and fingers domains. The result of functional assay indicated that MERS-CoV PL<sup>pro</sup> exhibits potent DUB activity but rather low proteolytic activity. A natural mutation, Leu105, is the major reason for this difference. The present study not only demonstrates the structural and functional characterization of MERS-CoV PL<sup>pro</sup>, but provides a foundation for further understanding the coronaviral PL<sup>pro</sup> family, which is an ideal antiviral target. Next, with pure protein and effective proteolytic activity assay, potent inhibitors of MERS-CoV PL<sup>pro</sup> can be high throughput screened and identified.</p></sec><sec sec-type="supplementary-material"><title>Electronic supplementary material</title><sec id="Sec16"><p><supplementary-material content-type="local-data" id="MOESM1"><media xlink:href="12929_2014_588_MOESM1_ESM.pdf"><caption><p>Additional file 1: Figure S1: Mass spectrometry of trypsin-digested peptides of the recombinant MERS-CoV PLpro protein. The red peaks show the signals of the peptides with correct mass, while the blue ones show the signals of the peptides with oxidation. X-axis indicates the m/z ratio and Y-axis shows the absorbance intensity. (PDF 101 KB)</p></caption></media></supplementary-material></p></sec></sec></body><back><app-group><app id="App1"><sec id="Sec17"><title>Authors’ original submitted files for images</title><p>Below are the links to the authors’ original submitted files for images.<media position="anchor" xlink:href="12929_2014_588_MOESM2_ESM.tif" id="MOESM2"><caption><p>Authors’ original file for figure 1</p></caption></media><media position="anchor" xlink:href="12929_2014_588_MOESM3_ESM.tif" id="MOESM3"><caption><p>Authors’ original file for figure 2</p></caption></media><media position="anchor" xlink:href="12929_2014_588_MOESM4_ESM.tif" id="MOESM4"><caption><p>Authors’ original file for figure 3</p></caption></media><media position="anchor" xlink:href="12929_2014_588_MOESM5_ESM.tif" id="MOESM5"><caption><p>Authors’ original file for figure 4</p></caption></media><media position="anchor" xlink:href="12929_2014_588_MOESM6_ESM.tif" id="MOESM6"><caption><p>Authors’ original file for figure 5</p></caption></media><media position="anchor" xlink:href="12929_2014_588_MOESM7_ESM.tif" id="MOESM7"><caption><p>Authors’ original file for figure 6</p></caption></media></p></sec></app></app-group><glossary><title>Abbreviations</title><def-list><def-item><term><sup>1</sup>AFC</term><def><p>7-amino-4-trifluoro–methylcoumarin</p></def></def-item><def-item><term>AUC</term><def><p>analytical ultracentrifugation</p></def></def-item><def-item><term>β-ME</term><def><p>β-mercaptoethanol</p></def></def-item><def-item><term>CD</term><def><p>circular dichroism</p></def></def-item><def-item><term>CoV</term><def><p>coronavirus</p></def></def-item><def-item><term>DUB</term><def><p>deubiquitinating protease</p></def></def-item><def-item><term>IRF3</term><def><p>interferon regulatory factor 3</p></def></def-item><def-item><term>MERS-CoV</term><def><p>Middle East respiratory syndrome coronavirus</p></def></def-item><def-item><term>PCR</term><def><p>polymerase chain reaction</p></def></def-item><def-item><term>PL<sup>pro</sup></term><def><p>papain-like protease</p></def></def-item><def-item><term>SARS-CoV</term><def><p>severe acute respiratory syndrome coronavirus</p></def></def-item><def-item><term>SV</term><def><p>sedimentation velocity</p></def></def-item><def-item><term>Ub</term><def><p>ubiquitin.</p></def></def-item></def-list></glossary><fn-group><fn><p><bold>Competing interests</bold></p><p>The authors declare that they have no competing interests.</p></fn><fn><p><bold>Authors’ contributions</bold></p><p>MHL carried out most experiments and analyzed the kinetic data. SJC expressed and purified the protein. CCC and CHL acquired and analyzed the data by mass spectrometry. SCC amplified the cDNA and constructed the expression plasmid. KWC participated in experimental design on structural analysis. CYS and CYC conceived the whole study, participated in experimental design and wrote the manuscript. All authors read and approved the final manuscript.</p></fn></fn-group><ack><title>Acknowledgements</title><p>This research was supported by grants from National Science Council, Taiwan (98-2320-B-010-026-MY3 and 101-2320-B-010-061) to CYC and CGMH-NYMU Joint Research Grant (CMRPG2D0211) to CYC and CYS. 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