Macro Domain from Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Is an Efficient ADP-ribose Binding Module
Identifieur interne : 000D80 ( Pmc/Corpus ); précédent : 000D79; suivant : 000D81Macro Domain from Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Is an Efficient ADP-ribose Binding Module
Auteurs : Chao-Cheng Cho ; Meng-Hsuan Lin ; Chien-Ying Chuang ; Chun-Hua HsuSource :
- The Journal of Biological Chemistry [ 0021-9258 ] ; 2016.
Abstract
The newly emerging Middle East respiratory syndrome coronavirus (MERS-CoV) encodes the conserved macro domain within non-structural protein 3. However, the precise biochemical function and structure of the macro domain is unclear. Using differential scanning fluorimetry and isothermal titration calorimetry, we characterized the MERS-CoV macro domain as a more efficient adenosine diphosphate (ADP)-ribose binding module than macro domains from other CoVs. Furthermore, the crystal structure of the MERS-CoV macro domain was determined at 1.43-Å resolution in complex with ADP-ribose. Comparison of macro domains from MERS-CoV and other human CoVs revealed structural differences in the α1 helix alters how the conserved Asp-20 interacts with ADP-ribose and may explain the efficient binding of the MERS-CoV macro domain to ADP-ribose. This study provides structural and biophysical bases to further evaluate the role of the MERS-CoV macro domain in the host response via ADP-ribose binding but also as a potential target for drug design.
Url:
DOI: 10.1074/jbc.M115.700542
PubMed: 26740631
PubMed Central: 4777827
Links to Exploration step
PMC:4777827Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Macro Domain from Middle East Respiratory Syndrome Coronavirus
(MERS-CoV) Is an Efficient ADP-ribose Binding Module</title><author><name sortKey="Cho, Chao Cheng" sort="Cho, Chao Cheng" uniqKey="Cho C" first="Chao-Cheng" last="Cho">Chao-Cheng Cho</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Lin, Meng Hsuan" sort="Lin, Meng Hsuan" uniqKey="Lin M" first="Meng-Hsuan" last="Lin">Meng-Hsuan Lin</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Chuang, Chien Ying" sort="Chuang, Chien Ying" uniqKey="Chuang C" first="Chien-Ying" last="Chuang">Chien-Ying Chuang</name><affiliation><nlm:aff id="aff2"></nlm:aff></affiliation><affiliation><nlm:aff id="aff3"></nlm:aff></affiliation></author><author><name sortKey="Hsu, Chun Hua" sort="Hsu, Chun Hua" uniqKey="Hsu C" first="Chun-Hua" last="Hsu">Chun-Hua Hsu</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation><affiliation><nlm:aff id="aff2"></nlm:aff></affiliation><affiliation><nlm:aff id="aff3"></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">26740631</idno><idno type="pmc">4777827</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4777827</idno><idno type="RBID">PMC:4777827</idno><idno type="doi">10.1074/jbc.M115.700542</idno><date when="2016">2016</date><idno type="wicri:Area/Pmc/Corpus">000D80</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000D80</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Macro Domain from Middle East Respiratory Syndrome Coronavirus
(MERS-CoV) Is an Efficient ADP-ribose Binding Module</title><author><name sortKey="Cho, Chao Cheng" sort="Cho, Chao Cheng" uniqKey="Cho C" first="Chao-Cheng" last="Cho">Chao-Cheng Cho</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Lin, Meng Hsuan" sort="Lin, Meng Hsuan" uniqKey="Lin M" first="Meng-Hsuan" last="Lin">Meng-Hsuan Lin</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Chuang, Chien Ying" sort="Chuang, Chien Ying" uniqKey="Chuang C" first="Chien-Ying" last="Chuang">Chien-Ying Chuang</name><affiliation><nlm:aff id="aff2"></nlm:aff></affiliation><affiliation><nlm:aff id="aff3"></nlm:aff></affiliation></author><author><name sortKey="Hsu, Chun Hua" sort="Hsu, Chun Hua" uniqKey="Hsu C" first="Chun-Hua" last="Hsu">Chun-Hua Hsu</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation><affiliation><nlm:aff id="aff2"></nlm:aff></affiliation><affiliation><nlm:aff id="aff3"></nlm:aff></affiliation></author></analytic><series><title level="j">The Journal of Biological Chemistry</title><idno type="ISSN">0021-9258</idno><idno type="eISSN">1083-351X</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>The newly emerging Middle East respiratory syndrome coronavirus (MERS-CoV)
encodes the conserved macro domain within non-structural protein 3. However, the
precise biochemical function and structure of the macro domain is unclear. Using
differential scanning fluorimetry and isothermal titration calorimetry, we
characterized the MERS-CoV macro domain as a more efficient adenosine
diphosphate (ADP)-ribose binding module than macro domains from other CoVs.
Furthermore, the crystal structure of the MERS-CoV macro domain was determined
at 1.43-Å resolution in complex with ADP-ribose. Comparison of macro
domains from MERS-CoV and other human CoVs revealed structural differences in
the α1 helix alters how the conserved Asp-20 interacts with ADP-ribose and
may explain the efficient binding of the MERS-CoV macro domain to ADP-ribose.
This study provides structural and biophysical bases to further evaluate the
role of the MERS-CoV macro domain in the host response via ADP-ribose binding
but also as a potential target for drug design.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Hui, D X0a S" uniqKey="Hui D">D.
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<front><journal-meta><journal-id journal-id-type="nlm-ta">J Biol Chem</journal-id><journal-id journal-id-type="iso-abbrev">J. Biol. Chem</journal-id><journal-id journal-id-type="hwp">jbc</journal-id><journal-id journal-id-type="pmc">jbc</journal-id><journal-id journal-id-type="publisher-id">JBC</journal-id><journal-title-group><journal-title>The Journal of Biological Chemistry</journal-title></journal-title-group><issn pub-type="ppub">0021-9258</issn><issn pub-type="epub">1083-351X</issn><publisher><publisher-name>American Society for Biochemistry and Molecular
Biology</publisher-name><publisher-loc>11200 Rockville Pike, Suite 302, Rockville, MD 20852-3110,
U.S.A.</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">26740631</article-id><article-id pub-id-type="pmc">4777827</article-id><article-id pub-id-type="publisher-id">M115.700542</article-id><article-id pub-id-type="doi">10.1074/jbc.M115.700542</article-id><article-categories><subj-group subj-group-type="heading"><subject>Protein Structure and Folding</subject></subj-group></article-categories><title-group><article-title>Macro Domain from Middle East Respiratory Syndrome Coronavirus
(MERS-CoV) Is an Efficient ADP-ribose Binding Module</article-title><subtitle>CRYSTAL STRUCTURE AND BIOCHEMICAL STUDIES<xref ref-type="fn" rid="FN1">*</xref></subtitle><alt-title alt-title-type="short">Structure of MERS-CoV Macro Domain</alt-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Cho</surname><given-names>Chao-Cheng</given-names></name><xref ref-type="aff" rid="aff1"><sup>‡</sup></xref></contrib><contrib contrib-type="author"><name><surname>Lin</surname><given-names>Meng-Hsuan</given-names></name><xref ref-type="aff" rid="aff1"><sup>‡</sup></xref></contrib><contrib contrib-type="author"><name><surname>Chuang</surname><given-names>Chien-Ying</given-names></name><xref ref-type="aff" rid="aff2"><sup>§</sup></xref><xref ref-type="aff" rid="aff3"><sup>¶</sup></xref></contrib><contrib contrib-type="author"><name><surname>Hsu</surname><given-names>Chun-Hua</given-names></name><xref ref-type="aff" rid="aff1"><sup>‡</sup></xref><xref ref-type="aff" rid="aff2"><sup>§</sup></xref><xref ref-type="aff" rid="aff3"><sup>¶</sup></xref><xref ref-type="corresp" rid="cor1"><sup>1</sup></xref></contrib><aff id="aff1">From the<label>‡</label>Genome and Systems Biology Degree Program, National Taiwan University and Academia Sinica, Taipei 10617,</aff><aff id="aff2">the<label>§</label>Department of Agricultural Chemistry, National Taiwan University, Taipei 10617, and</aff><aff id="aff3">the<label>¶</label>Center for Systems Biology, National Taiwan University, Taipei 10617, Taiwan</aff></contrib-group><author-notes><corresp id="cor1"><label>1</label> To whom correspondence should be addressed:
<addr-line>Dept. of Agricultural Chemistry, National Taiwan University,
Taipei 10617, Taiwan.</addr-line> Tel.: <phone>886-2-3366-4468</phone>; Fax:
<fax>886-2-3366-4468</fax>; E-mail:
<email>andyhsu@ntu.edu.tw</email>.</corresp></author-notes><pub-date pub-type="ppub"><day>4</day><month>3</month><year>2016</year></pub-date><pub-date pub-type="epub"><day>5</day><month>1</month><year>2016</year></pub-date><volume>291</volume><issue>10</issue><fpage>4894</fpage><lpage>4902</lpage><history><date date-type="received"><day>1</day><month>11</month><year>2015</year></date><date date-type="rev-recd"><day>28</day><month>12</month><year>2015</year></date></history><permissions><copyright-statement>© 2016 by The American Society for Biochemistry and
Molecular Biology, Inc.</copyright-statement><copyright-year>2016</copyright-year><copyright-holder>The American Society for Biochemistry and Molecular Biology,
Inc.</copyright-holder><license><license-p>This article is made available via the PMC Open Access Subset for
unrestricted re-use and analyses in any form or by any means with
acknowledgement of the original source. These permissions are granted for
the duration of the COVID-19 pandemic or until permissions are revoked in
writing. Upon expiration of these permissions, PMC is granted a perpetual
license to make this article available via PMC and Europe PMC, consistent
with existing copyright protections.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="zbc01016004894.pdf"></self-uri><abstract><p>The newly emerging Middle East respiratory syndrome coronavirus (MERS-CoV)
encodes the conserved macro domain within non-structural protein 3. However, the
precise biochemical function and structure of the macro domain is unclear. Using
differential scanning fluorimetry and isothermal titration calorimetry, we
characterized the MERS-CoV macro domain as a more efficient adenosine
diphosphate (ADP)-ribose binding module than macro domains from other CoVs.
Furthermore, the crystal structure of the MERS-CoV macro domain was determined
at 1.43-Å resolution in complex with ADP-ribose. Comparison of macro
domains from MERS-CoV and other human CoVs revealed structural differences in
the α1 helix alters how the conserved Asp-20 interacts with ADP-ribose and
may explain the efficient binding of the MERS-CoV macro domain to ADP-ribose.
This study provides structural and biophysical bases to further evaluate the
role of the MERS-CoV macro domain in the host response via ADP-ribose binding
but also as a potential target for drug design.</p></abstract><kwd-group><kwd>ADP-ribosylation</kwd><kwd>biophysics</kwd><kwd>crystal structure</kwd><kwd>RNA virus</kwd><kwd>viral protein</kwd></kwd-group><funding-group><award-group id="award1"><funding-source><institution-wrap><institution>Ministry of Science and Technology, Taiwan </institution><institution-id institution-id-type="open-funder-registry">10.13039/501100004663</institution-id></institution-wrap></funding-source><award-id>103–2113-M-002–009-MY2</award-id></award-group><award-group id="award2"><funding-source><institution-wrap><institution>National Taiwan University </institution><institution-id institution-id-type="open-funder-registry">10.13039/501100006477</institution-id></institution-wrap></funding-source><award-id>NTU-ERP-104R8600</award-id><award-id>NTU-ICRP-104R7560–5</award-id></award-group></funding-group></article-meta></front><body><sec sec-type="intro"><title>Introduction</title><p>Since the severe acute respiratory syndrome (SARS)<xref ref-type="fn" rid="FN2"><sup>2</sup></xref> outbreak in 2003 (<xref rid="B1" ref-type="bibr">1</xref>, <xref rid="B2" ref-type="bibr">2</xref>), a newly discovered disease,
Middle East respiratory syndrome (MERS), has been spreading from countries in the
Middle East to America (<xref rid="B3" ref-type="bibr">3</xref><xref ref-type="bibr" rid="B4">–</xref><xref rid="B5" ref-type="bibr">5</xref>). In the summer
of 2015, MERS was reported in North East Asia (<xref rid="B6" ref-type="bibr">6</xref><xref ref-type="bibr" rid="B7">–</xref><xref rid="B8" ref-type="bibr">8</xref>). The causative agent of MERS was identified as an unknown
coronavirus (CoV) resembling SARS-CoV and referred to as Middle East respiratory
syndrome CoV (MERS-CoV) (<xref rid="B9" ref-type="bibr">9</xref><xref ref-type="bibr" rid="B10">–</xref><xref rid="B12" ref-type="bibr">12</xref>). MERS-CoV belongs to the genus <italic>Betacoronavirus</italic> and
possesses a positive-strand RNA genome that encodes viral proteins essential to the
life cycle of the virus (<xref rid="B13" ref-type="bibr">13</xref>, <xref rid="B14" ref-type="bibr">14</xref>). The mortality of MERS is 4-fold higher
than SARS (40% compared with 10%) (<xref rid="B15" ref-type="bibr">15</xref>). Since
the first case report in Saudi Arabia, MERS has been reported in more than 20
countries and has caused more than 400 deaths worldwide (<xref rid="B9" ref-type="bibr">9</xref>).</p><p>CoVs utilize the RNA genome to encode structural proteins, including spike
glycoprotein (S), membrane protein (M), and nucleocapsid protein (N). They encode a
large number of non-structural proteins (NSPs) for rapid replication. A single large
replicase gene encodes all proteins involved in viral replication. The replicase
gene contains two open reading frames (ORFs), ORF1a and ORF1b, which encode two
polyproteins, pp1a and pp1ab; production of pp1ab requires a ribosomal frameshift to
transcribe the portion encoded by ORF1b (<xref rid="B16" ref-type="bibr">16</xref>).
ORF1a encodes viral proteases, main protease (M<sup>pro</sup>, also called
3CL<sup>pro</sup>), and papain-like protease (PL<sup>pro</sup>), which are
responsible for cleavage of the ORF1a and ORF1b gene products to produce functional
NSPs.</p><p>In SARS-CoV, the largest NSP member, NSP3, is a multidomain protein containing the
following domains: N-terminal acidic domain, macro domain, SARS-unique domain,
PL<sup>pro</sup>, nucleic acid-binding domain, marker domain (G<sub>2</sub>M),
transmembrane domain, and Y-domain (<xref rid="B17" ref-type="bibr">17</xref>). The
MERS-CoV genome contains 16 NSPs (<xref ref-type="fig" rid="F1">Fig. 1</xref>);
except for 3CL<sup>pro</sup> and PL<sup>pro</sup> (<xref rid="B18" ref-type="bibr">18</xref>, <xref rid="B19" ref-type="bibr">19</xref>), most of the functional
domains within the NSP3 in MERS-CoV remain structurally uncharacterized.</p><fig id="F1" orientation="portrait" position="float"><label>FIGURE 1.</label><caption><p><bold>Genome organization of MERS-CoV.</bold> Schematic diagram of the
composition of structural and non-structural proteins
(<italic>NSPs</italic>) in MERS-CoV genome. Functional domains of NSP3 are
highlighted. <italic>M</italic><sup>pro</sup>, main (or 3CL) protease;
<italic>RdRp</italic>, RNA-dependent RNA polymerase;
<italic>Hel</italic>, helicase; <italic>ExoN</italic>, exoribonuclease;
<italic>NendoU</italic>, endoribonuclease; <italic>OMT</italic>,
2′-O-methyltransferase; <italic>S</italic>, spike protein;
<italic>E</italic>, envelope protein; <italic>M</italic>, membrane
protein; <italic>N</italic>, nucleocapsid protein; <italic>MLS</italic>,
mitochondira localization signal; <italic>Macro</italic>, macro domain;
<italic>SUD-M</italic>, SARS-unique domain-M subdomain;
<italic>PL</italic><sup>pro</sup>, papain-like protease. NSPs encoded by
ORF1a and ORF1b are numbered in <italic>green</italic> and
<italic>blue</italic>, respectively.</p></caption><graphic xlink:href="zbc0111638450001"></graphic></fig><p>The macro domain is named after the non-histone motif of the histone variant
macroH2A, in which it was originally characterized (<xref rid="B20" ref-type="bibr">20</xref><xref ref-type="bibr" rid="B21">–</xref><xref rid="B22" ref-type="bibr">22</xref>), a protein module ubiquitous in eukaryotes, bacteria, and
archaea. This domain is well known for its affinity to adenosine diphosphate
(ADP)-ribose (<xref rid="B23" ref-type="bibr">23</xref><xref ref-type="bibr" rid="B24">–</xref><xref rid="B25" ref-type="bibr">25</xref>). Many
cellular enzymes bearing macro domains within their structures interact with
poly(ADP)-ribose (<xref rid="B26" ref-type="bibr">26</xref><xref ref-type="bibr" rid="B27">–</xref><xref rid="B29" ref-type="bibr">29</xref>).
Poly(ADP)-ribosylation is a post-translational modification linked with DNA repair,
apoptosis, gene regulation, and protein degradation. Thus, macro domain-containing
proteins and enzymes may play important roles in regulating various cellular
processes (<xref rid="B30" ref-type="bibr">30</xref>). Surprisingly, the CoVs
studied so far and a few other viruses such as alphavirus, rubella virus, and
hepatitis E virus possess macro domains in their genomes (<xref rid="B16" ref-type="bibr">16</xref>). In addition, some viral macro domains were found to have
ADP-ribose 1″-phosphate phosphatase (ADRP) activity (<xref rid="B31" ref-type="bibr">31</xref><xref ref-type="bibr" rid="B32">–</xref><xref rid="B33" ref-type="bibr">33</xref>), which catalyzes the removal of phosphate
from ADP-ribose 1″-phosphate (Appr1p) to produce ADP-ribose. ADRP activity
has been reported in a yeast protein containing macro domain as well as AF1521
protein in <italic>Archaeoglobus fulgidus</italic> (<xref rid="B23" ref-type="bibr">23</xref>, <xref rid="B34" ref-type="bibr">34</xref>, <xref rid="B35" ref-type="bibr">35</xref>). The enzymatic activity of viral macro domains in
processing Appr1p is low (<xref rid="B33" ref-type="bibr">33</xref>, <xref rid="B36" ref-type="bibr">36</xref><xref ref-type="bibr" rid="B37">–</xref><xref rid="B38" ref-type="bibr">38</xref>) and appears to be
dispensable for virus RNA synthesis (<xref rid="B31" ref-type="bibr">31</xref>). In
addition, the mutant for the CoV mouse hepatitis virus A59 (MHV-A59), encoding a
single amino acid substitution of a strictly conserved residue for ADRP activity,
replicated to slightly reduced titers in mouse liver but, strikingly, did not induce
liver disease (<xref rid="B39" ref-type="bibr">39</xref>). The MHV macro domain
exacerbates MHV-induced liver pathology, most likely by inducing excessive
inflammatory cytokine expression. It was also reported that catalytic residues
Asn-809, His-812, Gly-816, and Gly-817 for ADRP activity in hepatitis E virus macro
domain are critical for hepatitis E virus replication (<xref rid="B40" ref-type="bibr">40</xref>). Accordingly, the development of drugs targeting the viral
macro domain may be a strategy for antiviral therapy.</p><p>The macro domain of SARS-CoV NSP3 was previously reported to possess ADP-ribose and
poly(ADP)-ribose binding ability, which suggests that the macro domain may regulate
cellular proteins involved in an apoptotic pathway via poly(ADP)-ribosylation to
mediate the host response to infection (<xref rid="B36" ref-type="bibr">36</xref>).
Structural studies of macro domains from CoVs such as human CoV 229E (HCoV-229E) and
feline CoV (FCoV) also revealed interactions with ADP-ribose (<xref rid="B41" ref-type="bibr">41</xref><xref ref-type="bibr" rid="B42">–</xref><xref rid="B43" ref-type="bibr">43</xref>) and have offered huge advances in our
understanding of viral macro domains. The MERS-CoV genome features a macro domain
embedded in NSP3 (<xref ref-type="fig" rid="F1">Fig. 1</xref>). However, we lack
structural and functional information regarding the MERS-CoV macro domain.</p><p>In the present study, we investigated the MERS-CoV macro domain as an ADP-ribose
binding module, with comparison to previously characterized viral macro domains.
Furthermore, we determined the crystal structure of the MERS-CoV macro domain in
complex with ADP-ribose. Structural comparison of MERS-CoV and other human CoVs
revealed divergence in ADP-ribose binding by macro domains. Our study may shed new
light on structurally based design of novel antiviral drugs targeting viral macro
domains.</p></sec><sec sec-type="methods"><title>Experimental Procedures</title><sec><title></title><sec><title></title><sec><title>Protein Expression and Purification</title><p>The DNA sequence containing the MERS-CoV macro domain was synthesized by
a local biotechnology company (MDBio, Inc.) and cloned into the pUC57
plasmid. The macro domain fragment was inserted between the NdeI and
XhoI sites of the pET28a vector system (Novagen). The forward and
reverse PCR primers used for amplification were macro-F
(5′-AATTCATATGCCACTGAGCAATTTTGAACA-3′) and macro-R
(5′-AATTCTCGAGTTAGATGGTCAGGCTCTTATAC-3′). The resulting
plasmid with the inserted sequence was transformed into
<italic>Escherichia coli</italic> BL21(DE3) cells, which were grown
at 37 °C up to <italic>A</italic><sub>600</sub> 1.0 with 50
μg/ml of kanamycin. The expression of the recombinant MERS-CoV
macro domain with an His tag at the N terminus was induced in cells with
1 m<sc>m</sc> isopropyl β-<sc>d</sc>-thiogalactoside, followed by
growth for 20 h at 16 °C. Cells were collected by centrifugation
and resuspended in lysis buffer (25 m<sc>m</sc> phosphate buffer, pH
7.0, 100 m<sc>m</sc> NaCl). After 20 min of sonication, the cell extract
was clarified by centrifugation at 18,900 × <italic>g</italic> for
30 min at 4 °C to remove debris. The clear supernatant was placed
in an open column filled with nickel-nitrilotriacetic acid resin. The
resin was washed with 10 times volume of lysis buffer containing 50 and
100 m<sc>m</sc> imidazole, respectively. The His-tagged MERS-CoV macro
domain was eluted by lysis buffer containing 300 m<sc>m</sc> imidazole.
The purified MERS-CoV macro domain was dialyzed against stabilization
buffer (25 m<sc>m</sc> phosphate buffer, pH 7.0, 100 m<sc>m</sc> NaCl,
0.5 m<sc>m</sc> dithiothreitol). The His tag was removed by using
thrombin, which resulted in four additional residues (GSHM) at the N
terminus. The protein was further purified by gel filtration
chromatography with a Superdex75 XK 16/60 column (GE Healthcare) in 20
m<sc>m</sc> Tris-HCl buffer (pH 7.0), 100 m<sc>m</sc> NaCl.</p></sec><sec><title>Circular Dichroism (CD) Spectroscopy</title><p>Far-UV CD spectra were measured with 10 μ<sc>m</sc> protein samples
in CD buffer (20 m<sc>m</sc> phosphate buffer, pH 3.5–8.5) placed
into a 1-mm path length cuvette and recorded on a JASCO J-810
spectropolarimeter equipped with a Peltier temperature control system
(JASCO International Co.). Thermal transition of protein samples with or
without preincubation of 1 m<sc>m</sc> ADP-ribose were monitored at 220
nm from 25 to 95 °C at a scan rate of 1 °C/min. Baseline
subtraction, smoothing, and data normalization involved the use of
SigmaPlot. The melting temperature (<italic>T<sub>m</sub></italic>) was
calculated with the maximum of the first derivative of the CD
signal.</p></sec><sec><title>Differential Scanning Fluorimetry (DSF)</title><p>Thermal shift assay with DSF involved use of a CFX48 Real-time PCR
Detection System (Bio-Rad). In total, a 25-μl mixture containing 2
μl of SYPRO Orange (Sigma), 1.25 μl of dialysis buffer (20
m<sc>m</sc> Tris-HCl, and 100 m<sc>m</sc> NaCl, pH 7.0), 10 μl
of 1 μ<sc>m</sc> protein sample, and various concentrations of
ADP-ribose were mixed on ice in an 8-well PCR tube. Fluorescent signals
were measured from 25 to 95 °C in 0.1 °C/30-s steps
(excitation, 450–490 nm; detection, 560–580 nm). The main
measurements were carried out in triplicate. Data evaluation and
<italic>T<sub>m</sub></italic> determination involved use of the
Bio-Rad CFX Manager, and data fitting and dissociation constant
(<italic>K<sub>d</sub></italic>) calculations involved the use
of SigmaPlot.</p></sec><sec><title>Isothermal Titration Calorimetry (ITC)</title><p>Binding of ADP-ribose to the MERS-CoV macro domain was measured by ITC
with the Nano Isothermal Titration Calorimeter (TA Instruments).
Aliquots of 3 μl of 1.14 m<sc>m</sc> ADP-ribose were titrated by
injection into protein (0.057 m<sc>m</sc> in 0.98 ml) in 20 m<sc>m</sc>Tris-HCl (pH 7.0) and 100 m<sc>m</sc> NaCl. Experiments were carried out
at 25 °C with 250 rpm stirring. Background heat from ligand to
buffer titrations was subtracted, and the corrected heat from the
binding reaction was used to derive values for the stoichiometry of the
binding (<italic>n</italic>), <italic>K<sub>d</sub></italic>, apparent
enthalpy of binding (Δ<italic>H</italic>), and entropy change
(Δ<italic>S</italic>). Data were fitted by use of an
independent binding model with Launch NanoAnalyze version 2.3.6.</p></sec><sec><title>Crystallization and Data Collection</title><p>The MERS-CoV macro domain and ADP-ribose were mixed in a molar ratio of
1:15. Initial protein crystallization trials were performed at 283 K by
the sitting-drop vapor-diffusion method with commercial crystallization
screen kits, 96-well Intelli-plates (Art Robbins Instruments), and a
HoneyBee 963 robot (Genomic Solutions). Each crystallization drop was
prepared by mixing 0.3 μl of macro domain/ADP-ribose at 10 mg/ml
with an equal volume of mother liquor, and the mixture was equilibrated
against 100 μl of reservoir solution. The crystals for data
collection were grown in 1 week at 283 K with the optimal condition of
100 m<sc>m</sc> phosphate/citrate (pH 4.2), 2.0 <sc>m</sc> ammonium
sulfate, and 10 m<sc>m</sc> nicotinamide adenine dinucleotide as the
additive. For subsequent anomalous phasing, the crystal was soaked for 8
h in 3 m<sc>m</sc> mercuric(II) chloride, cryoprotected in mother liquor
supplemented with 20% glycerol, and flash-frozen in liquid nitrogen at
100 K. The diffraction images were recorded in a 100-K nitrogen gas
stream with use of BL13B1 or BL13C1 beamlines (National Synchrotron
Radiation Research Center, Taiwan) and processed by using HKL2000
software (<xref rid="B44" ref-type="bibr">44</xref>).</p></sec><sec><title>Structure Determination and Refinement</title><p>The crystal structure of the MERS-CoV macro domain in complex with
ADP-ribose was solved by the mercury(II) derivative single-wavelength
anomalous dispersion method by using SHELXD/SHELXE software (<xref rid="B45" ref-type="bibr">45</xref>). The initial model was refined
by the maximum likelihood method implemented in REFMAC5 (<xref rid="B46" ref-type="bibr">46</xref>) as part of the CCP4 suite
(<xref rid="B47" ref-type="bibr">47</xref>) and rebuilt
interactively by inspecting the σ-weighted electron density maps
with coefficients 2<italic>mF</italic><sub>o</sub> −
<italic>DF<sub>c</sub></italic> and
<italic>mF<sub>o</sub></italic> −
<italic>DF<sub>c</sub></italic> in COOT (<xref rid="B48" ref-type="bibr">48</xref>). During the later stages, restrained positional
and B-factor refinement involved the program phenix.refine (<xref rid="B49" ref-type="bibr">49</xref>). Water molecules were manually
added at the final stages. The models were evaluated with use of
PROCHECK (<xref rid="B50" ref-type="bibr">50</xref>) and MOLPROBITY
(<xref rid="B51" ref-type="bibr">51</xref>). The data collection and
structure refinement statistics are in <xref rid="T1" ref-type="table">Table 1</xref>.</p><table-wrap id="T1" orientation="portrait" position="float"><label>TABLE 1</label><caption><p><bold>Data collection and refinement statistics of MERS-CoV macro
domain in complex with ADP-ribose</bold></p></caption><table frame="hsides" rules="groups"><tbody valign="top"><tr><td align="left" rowspan="1" colspan="1"><bold>Crystal
parameters</bold></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"> Crystal</td><td align="left" rowspan="1" colspan="1">Hg-SAD</td><td align="left" rowspan="1" colspan="1">Native</td></tr><tr><td align="left" rowspan="1" colspan="1"> Space group</td><td align="left" rowspan="1" colspan="1"><italic>C</italic>222<sub>1</sub></td><td align="left" rowspan="1" colspan="1"><italic>C</italic>222<sub>1</sub></td></tr><tr><td align="left" rowspan="1" colspan="1"> Unit
cell parameters</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"> <italic>a</italic>,
<italic>b</italic>, <italic>c</italic> (Å)</td><td align="left" rowspan="1" colspan="1">41.4; 120.8;
66.7</td><td align="left" rowspan="1" colspan="1">41.8; 120.8;
67.7</td></tr><tr><td align="left" rowspan="1" colspan="1"> α,
β, γ (°)</td><td align="left" rowspan="1" colspan="1">90, 90, 90</td><td align="left" rowspan="1" colspan="1">90, 90, 90</td></tr><tr><td align="left" rowspan="1" colspan="1"> Monomers
per asymmetric unit cell</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">1</td></tr><tr><td colspan="3" rowspan="1"><hr></hr></td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>Data
collection</bold></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"> Wavelength
(Å)</td><td align="left" rowspan="1" colspan="1">0.99347</td><td align="left" rowspan="1" colspan="1">1.00545</td></tr><tr><td align="left" rowspan="1" colspan="1"> Resolution range
(Å)</td><td align="left" rowspan="1" colspan="1">26.51–1.73
(1.79–1.73)</td><td align="left" rowspan="1" colspan="1">22.53–1.43
(1.48–1.43)</td></tr><tr><td align="left" rowspan="1" colspan="1"> Unique No. of
reflections</td><td align="left" rowspan="1" colspan="1">17,591</td><td align="left" rowspan="1" colspan="1">31,889</td></tr><tr><td align="left" rowspan="1" colspan="1"> Total No. of
reflections</td><td align="left" rowspan="1" colspan="1">229,930</td><td align="left" rowspan="1" colspan="1">186,275</td></tr><tr><td align="left" rowspan="1" colspan="1"> <italic>I</italic>/σ<xref ref-type="table-fn" rid="TF1-1"><italic><sup>a</sup></italic></xref></td><td align="left" rowspan="1" colspan="1">43.7 (4.5)</td><td align="left" rowspan="1" colspan="1">37.2 (6.8)</td></tr><tr><td align="left" rowspan="1" colspan="1"> <italic>R</italic><sub>merge</sub><italic><sup><xref ref-type="table-fn" rid="TF1-1">a</xref>,<xref ref-type="table-fn" rid="TF1-2">b</xref></sup></italic> (%)</td><td align="left" rowspan="1" colspan="1">6.7 (49.1)</td><td align="left" rowspan="1" colspan="1">2.9 (25.1)</td></tr><tr><td align="left" rowspan="1" colspan="1"> Completeness<xref ref-type="table-fn" rid="TF1-1"><italic><sup>a</sup></italic></xref> (%)</td><td align="left" rowspan="1" colspan="1">99.1 (98.7)</td><td align="left" rowspan="1" colspan="1">99.8 (100.0)</td></tr><tr><td align="left" rowspan="1" colspan="1"> Redundancy<xref ref-type="table-fn" rid="TF1-1"><italic><sup>a</sup></italic></xref></td><td align="left" rowspan="1" colspan="1">13.1 (12.6)</td><td align="left" rowspan="1" colspan="1">5.8 (5.8)</td></tr><tr><td align="left" rowspan="1" colspan="1"> CC<sub>1/2</sub><italic><sup><xref ref-type="table-fn" rid="TF1-1">a</xref>,<xref ref-type="table-fn" rid="TF1-3">c</xref></sup></italic></td><td align="left" rowspan="1" colspan="1">0.989 (0.953)</td><td align="left" rowspan="1" colspan="1">0.993 (0.972)</td></tr><tr><td align="left" rowspan="1" colspan="1"> CC<sub>ano</sub><xref ref-type="table-fn" rid="TF1-4"><italic><sup>d</sup></italic></xref></td><td align="left" rowspan="1" colspan="1">0.63</td><td rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"> Anomalous
redundancy<xref ref-type="table-fn" rid="TF1-1"><italic><sup>a</sup></italic></xref></td><td align="left" rowspan="1" colspan="1">6.9 (6.6)</td><td rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"> Anomalous
completeness<xref ref-type="table-fn" rid="TF1-1"><italic><sup>a</sup></italic></xref> (%)</td><td align="left" rowspan="1" colspan="1">98.9 (98.6)</td><td rowspan="1" colspan="1"></td></tr><tr><td colspan="3" rowspan="1"><hr></hr></td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>Refinement
statistics</bold></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"> Resolution
(Å)</td><td rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">1.43</td></tr><tr><td align="left" rowspan="1" colspan="1"> <italic>R</italic><sub>work</sub>(%)/<italic>R</italic><sub>free</sub> (%)<xref ref-type="table-fn" rid="TF1-5"><italic><sup>e</sup></italic></xref></td><td rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">12.73 / 16.19</td></tr><tr><td align="left" rowspan="1" colspan="1"> R.m.s. deviation</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"> Bonds
(Å)</td><td rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">0.007</td></tr><tr><td align="left" rowspan="1" colspan="1"> Angles
(°)</td><td rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">1.213</td></tr><tr><td align="left" rowspan="1" colspan="1"> Mean B-factor
(Å<sup>2</sup>)</td><td rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">20.6</td></tr><tr><td align="left" rowspan="1" colspan="1"> Protein</td><td rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">17.2</td></tr><tr><td align="left" rowspan="1" colspan="1"> ADP-ribose</td><td rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">37.6</td></tr><tr><td align="left" rowspan="1" colspan="1"> Water</td><td rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">36.3</td></tr><tr><td align="left" rowspan="1" colspan="1"> Ramachandran plot
(%)</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"> Favored</td><td rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">93.8</td></tr><tr><td align="left" rowspan="1" colspan="1"> Allowed</td><td rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">6.2</td></tr><tr><td align="left" rowspan="1" colspan="1"> Outliers</td><td rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">0.0</td></tr></tbody></table><table-wrap-foot><fn id="TF1-1"><p><italic><sup>a</sup></italic> Values in parentheses are for
the highest resolution shell.</p></fn><fn id="TF1-2"><p><italic><sup>b</sup> R</italic><sub>merge</sub> =
Σ<italic><sub>h</sub></italic>Σ<italic><sub>i</sub></italic>|<italic>I<sub>h,i</sub></italic>−<italic>I<sub>h</sub></italic>|/Σ<italic><sub>h</sub></italic>Σ<italic><sub>i</sub>I<sub>h,i</sub></italic>,
where <italic>I<sub>h</sub></italic> is the mean intensity
of the <italic>i</italic> observations of symmetry related
reflections of <italic>h</italic>.</p></fn><fn id="TF1-3"><p><italic><sup>c</sup></italic> CC<sub>1/2</sub> is a
percentage of correlation between intensities from random
half-datasets (<xref rid="B56" ref-type="bibr">56</xref>).</p></fn><fn id="TF1-4"><p><italic><sup>d</sup></italic> CC<sub>ano</sub> is a
percentage of correlation between random half-datasets of
anomalous intensity differences.</p></fn><fn id="TF1-5"><p><italic><sup>e</sup>R</italic><sub>work</sub>/<italic>R</italic><sub>free</sub>= Σ|<italic>F</italic><sub>obs</sub> −
<italic>F</italic><sub>calc</sub>|/Σ<italic>F</italic><sub>obs</sub>,
where <italic>F</italic><sub>calc</sub> is the calculated
protein structure factor from the atomic model
(<italic>R</italic><sub>free</sub> was calculated with
5% of the reflections selected).</p></fn></table-wrap-foot></table-wrap></sec></sec></sec></sec><sec sec-type="results|discussion"><title>Results and Discussion</title><sec><title></title><sec><title></title><sec><title>ADP-ribose Binding Ability of MERS-CoV Macro Domain</title><p>The MERS-CoV macro domain (pp1a residues 1110 to 1273) was expressed and
purified from <italic>E. coli</italic>. The final purified protein was a
167-amino acid protein (20 kDa), with four additional residues at the N
terminus resulting from removal of the hexa-histidine tag after thrombin
cleavage. CD spectra revealed that the macro domain exhibited a stable
α/β-type folding pattern under various pH conditions (<xref ref-type="fig" rid="F2">Fig. 2</xref>). The
<italic>T<sub>m</sub></italic> of the macro domain from thermal
transition monitored by CD was 43 °C. However, the addition of
ADP-ribose significantly increased the <italic>T<sub>m</sub></italic> to
51 °C (<xref ref-type="fig" rid="F3">Fig.
3</xref><italic>A</italic>). The significant increase in
<italic>T<sub>m</sub></italic> suggests the interaction between
the MERS-CoV macro domain and ADP-ribose.</p><fig id="F2" orientation="portrait" position="float"><label>FIGURE 2.</label><caption><p><bold>Folding of MERS-CoV macro domain.</bold> The CD spectra
were recorded at 25 °C with 10 μ<sc>m</sc> MERS-CoV
macro domain in CD buffer (20 m<sc>m</sc> phosphate buffer, pH
3.5–8.5) from 260 to 190 nm.</p></caption><graphic xlink:href="zbc0111638450002"></graphic></fig><fig id="F3" orientation="portrait" position="float"><label>FIGURE 3.</label><caption><p><bold>ADP-ribose binding of MERS-CoV macro domain.</bold><italic>A,</italic> thermal denaturation of MERS-CoV macro
domain. CD spectra were recorded at 220 nm with 10
μ<sc>m</sc> MERS-CoV macro domain in CD buffer (20
m<sc>m</sc> phosphate buffer, pH 7.5) from 25 to 95 °C.
The scatterplot shows the MERS-CoV macro domain with and without
preincubation with 1 m<sc>m</sc> ADP-ribose, in
<italic>blue</italic> and <italic>red</italic>,
respectively. The melting temperature
(<italic>T<sub>m</sub></italic>) was calculated by using the
maximum of the first derivative of the CD signal; the
<italic>black arrow</italic> indicates the shift of
<italic>T<sub>m</sub>. B,</italic> differential scanning
fluorimetry of MERS-CoV macro domain by thermal shift assay on
incubation with increasing concentrations of ADP-ribose. Data
are mean ± S.E. from 3 independent experiments. Data were
fitted by the means of 3 independent experiments.
<italic>C,</italic> isothermal titration calorimetry
analysis of ADP-ribose binding to MERS-CoV macro domain.
<italic>Upper panel</italic>, raw data in μJ/s
<italic>versus</italic> time showing heat release on
injection of 1.14 m<sc>m</sc> ADP-ribose into a 980-μl
cell containing 0.057 m<sc>m</sc> MERS-CoV macro domain.
<italic>Lower panel</italic>, integration of raw data
yielding the heat per mole <italic>versus</italic> molar ratio.
The <italic>inset</italic> shows thermodynamic parameters of the
experiment.</p></caption><graphic xlink:href="zbc0111638450003"></graphic></fig><p>To understand the affinity of ADP-ribose binding to the MERS-CoV macro
domain, both DSF and ITC measurements were used to examine the
equilibrium dissociation constant (<italic>K<sub>d</sub></italic>) of
ADP-ribose. After fitting DSF data, the <italic>K<sub>d</sub></italic>was determined to be 3.12 ± 0.42 μ<sc>m</sc>(<italic>r</italic><sup>2</sup> = 0.9628) (<xref ref-type="fig" rid="F3">Fig. 3</xref><italic>B</italic>), which is similar to the
calculated <italic>K<sub>d</sub></italic> of 2.95 μ<sc>m</sc>based on ITC data (<xref ref-type="fig" rid="F3">Fig.
3</xref><italic>C</italic>). In addition, ITC data indicated that
ADP-ribose bound to the MERS-CoV macro domain with favorable enthalpy
change (exothermic, Δ<italic>H</italic> = −91.04 KJ/mol).
The binding reaction was spontaneous at 25 °C with exergonic Gibbs
energy of binding (Δ<italic>G</italic> = −31.56 KJ/mol).
The thermodynamic profile (Δ<italic>G</italic> < 0,
Δ<italic>H</italic> < 0, and
−<italic>T</italic>Δ<italic>S</italic> > 0) of
ADP-ribose binding to the MERS-CoV macro domain suggests that ADP-ribose
is likely stabilized by hydrogen bond formations (<xref rid="B52" ref-type="bibr">52</xref>).</p><p>We reviewed the results of previously reported binding assays of
ADP-ribose binding to CoV macro domains (<xref rid="T2" ref-type="table">Table 2</xref>). Compared with the <italic>K<sub>d</sub></italic>of ADP-ribose binding to macro domains of human CoVs such as SARS-CoV
(24 μ<sc>m</sc>) (<xref rid="B36" ref-type="bibr">36</xref>) and
HCoV-229E (28.9 μ<sc>m</sc>) (<xref rid="B41" ref-type="bibr">41</xref>) and animal coronaviruses such as FCoV (∼400
μ<sc>m</sc>) (<xref rid="B42" ref-type="bibr">42</xref>), our
<italic>K<sub>d</sub></italic> of 2.95 μ<sc>m</sc> from
biochemical analysis suggests that the MERS-CoV macro domain is a more
efficient ADP-ribose binding module. The SARS macro domain possesses
poly(ADP)-ribose binding ability and may play a role in the host
response to virus (<xref rid="B36" ref-type="bibr">36</xref>). We found
that the MERS-CoV macro domain interacts with ADP-ribose, which suggests
further investigating the role of the macro domain in MERS-CoV
infection.</p><table-wrap id="T2" orientation="portrait" position="float"><label>TABLE 2</label><caption><p><bold>Binding assays of ADP-ribose in CoV macro
domains</bold></p></caption><table frame="hsides" rules="groups"><thead valign="bottom"><tr><th align="center" rowspan="1" colspan="1">Virus</th><th align="center" rowspan="1" colspan="1">Method</th><th align="center" rowspan="1" colspan="1"><italic>K<sub>d</sub></italic></th><th align="center" rowspan="1" colspan="1">Δ<italic>H</italic></th><th align="center" rowspan="1" colspan="1">Δ<italic>S</italic></th><th align="center" rowspan="1" colspan="1">−<italic>T</italic>Δ<italic>S</italic></th><th align="center" rowspan="1" colspan="1">Δ<italic>G</italic></th><th align="center" rowspan="1" colspan="1">References</th></tr></thead><tbody valign="top"><tr><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">μ<italic><sc>m</sc></italic></td><td align="left" rowspan="1" colspan="1">KJ/mol</td><td align="left" rowspan="1" colspan="1">J/mol·K</td><td align="left" rowspan="1" colspan="2">KJ/mol</td><td rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="2" colspan="1">MERS-CoV</td><td align="left" rowspan="1" colspan="1">ITC</td><td align="left" rowspan="1" colspan="1">2.95</td><td align="left" rowspan="1" colspan="1">−91.04</td><td align="left" rowspan="1" colspan="1">−199.5</td><td align="left" rowspan="1" colspan="1">59.48</td><td align="left" rowspan="1" colspan="1">−31.56</td><td align="left" rowspan="1" colspan="1">This study</td></tr><tr><td align="left" rowspan="1" colspan="1">DSF</td><td align="left" rowspan="1" colspan="1">3.12</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">This study</td></tr><tr><td align="left" rowspan="1" colspan="1">SARS-CoV</td><td align="left" rowspan="1" colspan="1">ITC</td><td align="left" rowspan="1" colspan="1">24</td><td align="left" rowspan="1" colspan="1">−73.39</td><td align="left" rowspan="1" colspan="1">−153.9</td><td align="left" rowspan="1" colspan="1">46.65</td><td align="left" rowspan="1" colspan="1">−26.74</td><td align="left" rowspan="1" colspan="1"><xref rid="B36" ref-type="bibr">36</xref></td></tr><tr><td align="left" rowspan="1" colspan="1">HCoV-229E</td><td align="left" rowspan="1" colspan="1">ITC</td><td align="left" rowspan="1" colspan="1">28.9</td><td align="left" rowspan="1" colspan="1">−14.54</td><td align="left" rowspan="1" colspan="1">38.1</td><td align="left" rowspan="1" colspan="1">−11.36</td><td align="left" rowspan="1" colspan="1">−25.9</td><td align="left" rowspan="1" colspan="1"><xref rid="B41" ref-type="bibr">41</xref></td></tr><tr><td align="left" rowspan="1" colspan="1">FCoV</td><td align="left" rowspan="1" colspan="1">Pull-down based
binding assay</td><td align="left" rowspan="1" colspan="1">∼400</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"><xref rid="B42" ref-type="bibr">42</xref></td></tr></tbody></table></table-wrap></sec><sec><title>Overall Structure of MERS-CoV Macro Domain in Complex with
ADP-ribose</title><p>We determined the crystal structure of ADP-ribose-bound MERS-CoV macro
domain for further molecular elucidation. The orthorhombic crystals gave
good quality x-ray diffraction and belonged to the space group
C222<sub>1</sub> with the following unit cell dimensions:
<italic>a</italic> = 41.798 Å, <italic>b</italic> = 120.807
Å, <italic>c</italic> = 67.659 Å, and α = β =
γ = 90°. The structure of the MERS-CoV macro domain was
solved by mercury single-wavelength anomalous dispersion (see
“Experimental Procedures”). The final protein structure
(<xref ref-type="fig" rid="F4">Fig. 4</xref><italic>A</italic>) was
refined to 1.43-Å resolution with <italic>R</italic>-factor and
<italic>R</italic>-free values of 0.1273 and 0.1619, respectively
(<xref rid="T1" ref-type="table">Table 1</xref>). The core of the
structure of MERS-CoV macro domain is a seven-stranded β-sheet in
the order of
β1-β2-β7-β6-β3-β5-β4 (<xref ref-type="fig" rid="F4">Fig. 4</xref><italic>B</italic>). The
central β-sheet is sandwiched between six α-helices, with
α1, α2, and α3 packing onto one face and α4,
α5, and α6 onto the other. In the initial refinement cycle,
a strong bent electron density (continuous at 1σ cutoff) (<xref ref-type="fig" rid="F4">Fig. 4</xref><italic>A</italic>) located at
the central pocket, was unambiguously identified as an ADP-ribose
molecule. This molecule is tightly bound in an uncharged crevice located
at the C-terminal end of strands β3 and β6 in the loop
regions between β3-α2 and β6-α5 (<xref ref-type="fig" rid="F4">Fig. 4</xref><italic>A</italic>).</p><fig id="F4" orientation="portrait" position="float"><label>FIGURE 4.</label><caption><p><bold>Overall structure of MERS-CoV macro domain in complex with
ADP-ribose.</bold><italic>A,</italic> structure of the MERS-CoV macro domain is
represented by a ribbon model with helices, strands, and loops
in <italic>magenta</italic>, <italic>yellow</italic>, and
<italic>blue</italic>, respectively. ADP-ribose is displayed
in sticks with carbon in <italic>green</italic>, oxygen in
<italic>red</italic>, nitrogen in <italic>blue</italic>, and
phosphorus in <italic>orange</italic>. The
2<italic>F<sub>o</sub></italic> −
<italic>F<sub>c</sub></italic> difference map, contoured
at 1σ, was calculated at 1.43-Å resolution from a
model with the ligand omitted. <italic>B</italic>, topology
diagram of MERS-CoV macro domain with the same colors as with
ribbon representation.</p></caption><graphic xlink:href="zbc0111638450004"></graphic></fig><p>A search of the DALI database (<xref rid="B53" ref-type="bibr">53</xref>)
with the structure of the MERS-CoV macro domain in complex with
ADP-ribose used as a model revealed several structural homologs.
Top-ranked structures were macro domains of CoVs in complex with
ADP-ribose such as those for SARS-CoV (PDB code <ext-link ext-link-type="pdb" xlink:href="2FAV">2FAV</ext-link>; <italic>Z</italic> score 27.9;
r.m.s. deviation 1.3; sequence identity 45%; sequence similarity 65%)
(<xref rid="B36" ref-type="bibr">36</xref>), HCoV-229E (PDB code
<ext-link ext-link-type="pdb" xlink:href="3EWR">3EWR</ext-link>; <italic>Z</italic> score 22.8; r.m.s. deviation
1.8; sequence identity 33%; sequence similarity 56%) (<xref rid="B43" ref-type="bibr">43</xref>), FCoV (PDB code <ext-link ext-link-type="pdb" xlink:href="3JZT">3JZT</ext-link>, Z score 22.6; r.m.s. deviation
1.8; sequence identity 30%; sequence similarity 53%) (<xref rid="B42" ref-type="bibr">42</xref>), and infectious bronchitis
virus (PDB code <ext-link ext-link-type="pdb" xlink:href="3EWP">3EWP</ext-link>; Z score 19.5; r.m.s. deviation 1.9; sequence
identity 28%; sequence similarity 47%) (<xref rid="B43" ref-type="bibr">43</xref>). This finding reflects that the viral macro domains are
structurally well conserved. However, variability between all these
structures arises from the loops connecting the core secondary structure
elements, which display great diversity in sequence, length, and
conformation and may correspond to different ADP-ribose binding
ability.</p></sec><sec><title>Molecular Basis of ADP-ribose Binding in MERS-CoV Macro
Domain</title><p>To gain insights into the molecular mechanism of ADP-ribose binding, we
further investigated the binding pocket for ADP-ribose in the MERS-CoV
macro domain. The adenine moiety resides in the hydrophobic cavity
containing Gly-19, Ala-21, Ile-47, Pro-123, Leu-124, and Val-152 (<xref ref-type="fig" rid="F5">Fig. 5</xref><italic>A</italic>).
Coordination of ADP-ribose involves serial hydrogen bond formations and
hydrophobic interactions provided by surrounding amino acid residues
(<xref ref-type="fig" rid="F5">Fig. 5</xref><italic>B</italic>). The
side chain of Asp-20 contacts the N-6 atom of the pyrimidine ring in
adenine moiety via direct hydrogen bonding. This residue is critical for
binding specificity of the macro domain AF1521 in <italic>A.
fulgidus</italic> (<xref rid="B54" ref-type="bibr">54</xref>).
Structure-based multiple sequence alignment showed that this aspartic
acid is conserved among CoV macro domains (<xref ref-type="fig" rid="F6">Fig. 6</xref><italic>A</italic>). Oxygen atoms of the pyrophosphate
in ADP-ribose contact surrounding residues via hydrogen bonding with
nitrogen atoms in backbone amides of Ile-47, Ser-126, Gly-128, Ile-129,
and Phe-130. The second ribose is stabilized by complex hydrogen bonding
with surrounding residues and water molecules (<xref ref-type="fig" rid="F5">Fig. 5</xref><italic>A</italic>). The ribose-3″
oxygen atom forms a hydrogen bond with a nitrogen atom in the side chain
of Asn-38. The ribose-2″ oxygen atom forms hydrogen bonds with
the oxygen and nitrogen atoms in the backbone amides of Lys-42 and
Gly-44, respectively. The ribose-1″ oxygen atom forms a hydrogen
bond with the nitrogen atom in the backbone amide of Gly-46. A water
molecule serves as a bridge between the ribose-1″ oxygen atom,
Asn-38, and His-43. This organization of the terminal ribose and
surrounding molecules was also observed in the yeast ADRP enzyme (<xref rid="B34" ref-type="bibr">34</xref>), which suggests that Asn-38 and
His-43 may be critical for the hydrolysis reaction of ADP-ribose
1″-phosphate to ADP-ribose. In addition, equivalent residues
critical for ADRP activity in the SARS-CoV macro domain (<xref rid="B36" ref-type="bibr">36</xref>) included Asn-35, Asn-38,
His-43, Gly-44, Gly-45, and Phe-130, which are conserved in the MERS-CoV
macro domain (<xref ref-type="fig" rid="F6">Fig.
6</xref><italic>A</italic>). Conservation of catalytically
significant residues of ADRP in the MERS-CoV macro domain indicates that
the MERS-CoV macro domain may possess ADRP enzymatic activity.</p><fig id="F5" orientation="portrait" position="float"><label>FIGURE 5.</label><caption><p><bold>Detailed view of ADP-ribose binding site in MERS-CoV macro
domain.</bold><italic>A</italic>, a close-up of interactions in MERS-CoV macro
domain with ADP-ribose binding. Amino acids and ADP-ribose are
shown as sticks with carbon in <italic>marine blue</italic> and
<italic>yellow</italic>, respectively; oxygen in
<italic>red</italic>; nitrogen in <italic>blue</italic>; and
phosphorus in orange. Water molecules are shown as <italic>green
spheres</italic>. Hydrogen bonds are <italic>black dashed
lines. B,</italic> interactions between MERS-CoV macro
domain and ADP-ribose. Interactions between MERS-CoV macro
domain and ADP-ribose were generated by using
LigPlot<sup>+</sup> (<xref rid="B55" ref-type="bibr">55</xref>). ADP-ribose and surrounding residues are shown
as ball-and-stick models with carbon in <italic>black</italic>,
nitrogen in <italic>blue</italic>, oxygen in
<italic>red</italic>, and phosphorus in
<italic>purple</italic>. Atomic bonds in ADP-ribose and the
MERS-CoV macro domain are in <italic>purple</italic> and
<italic>yellow</italic>, respectively. Residues contacting
ADP-ribose via hydrogen bonds are highlighted in
<italic>green</italic> with hydrogen bonds shown as
<italic>dashed lines</italic> and bond length as
<italic>numeric numbers</italic>. Residues that provide
hydrophobic interactions with ADP-ribose are in
<italic>black</italic> with <italic>red eyelash
symbols</italic>.</p></caption><graphic xlink:href="zbc0111638450005"></graphic></fig><fig id="F6" orientation="portrait" position="float"><label>FIGURE 6.</label><caption><p><bold>Structural comparison of MERS-CoV, SARS-CoV, and HCoV-229E
macro domains.</bold><italic>A,</italic> structure-based sequence alignment of CoV
macro domains. Shown are MERS-CoV (PDB code <ext-link ext-link-type="pdb" xlink:href="5DUS">5DUS</ext-link>); SARS-CoV (PDB code <ext-link ext-link-type="pdb" xlink:href="2FAV">2FAV</ext-link>); human coronavirus 229E (HCoV-229E; PDB
code <ext-link ext-link-type="pdb" xlink:href="3EWR">3EWR</ext-link>); HCoV-NL63 (PDB code <ext-link ext-link-type="pdb" xlink:href="2VRI">2VRI</ext-link>); and feline CoV (FCoV; PDB code <ext-link ext-link-type="pdb" xlink:href="3JZT">3JZT</ext-link>); and infectious bronchitis virus
(<italic>IBV</italic>; PDB code <ext-link ext-link-type="pdb" xlink:href="3EWP">3EWP</ext-link>). Secondary structures of MERS-CoV macro
domain are depicted on the <italic>top</italic> of the alignment
with <italic>arrows</italic> for β strands and
<italic>cylinders</italic> for α helices. Consensus
amino acids among macro domains in CoVs with similarity score
>0.7 are framed in <italic>yellow</italic> and depicted at
the <italic>bottom</italic> of the alignment. Identical amino
acids are in <italic>white</italic> and framed in <italic>red.
Blue</italic> and <italic>green arrowheads</italic> on the
top indicate amino acids forming hydrogen bonds and providing
hydrophobic interactions with ADP-ribose, respectively.
<italic>Yellow arrowheads</italic> at the bottom indicate
equivalent amino acids in SARS-CoV macro domain found to abolish
or decrease ADRP enzymatic activities when mutated. The number
of residues corresponding to the MERS-CoV macro domain indicated
by <italic>blue</italic>, <italic>green</italic>, and
<italic>yellow arrowheads</italic> is shown on the top of
the alignment. <italic>B,</italic> superposition of macro
domains. Structures are shown as a ribbon model with MERS-CoV in
<italic>blue</italic>, SARS-CoV in <italic>pink</italic>,
and HCoV-229E in <italic>green</italic>. ADP-ribose molecules
are shown as a stick model. Structural divergence is
<italic>circled with a black oval. C,</italic> comparison of
interactions in the adenine cavity of MERS-CoV and SARS-CoV
macro domains. Amino acids and ADP-ribose are shown as a stick
model. Hydrogen bonds are shown as <italic>dashed lines</italic>and bond lengths are indicated in Å units.
<italic>D,</italic> comparison of interactions in adenine
cavities of MERS-CoV and HCoV-229E macro domains. Amino acids
and ADP-ribose are shown as a <italic>stick model</italic>.
Hydrogen bonds are shown as <italic>dashed lines</italic>.</p></caption><graphic xlink:href="zbc0111638450006"></graphic></fig></sec><sec><title>Structural Comparison of Macro Domains in MERS-CoV, SARS-CoV, and
HCoV-229E</title><p>The structures of the macro domains of other CoVs pathogenic to humans,
including SARS-CoV (<xref rid="B36" ref-type="bibr">36</xref>) and
HCoV-229E (<xref rid="B43" ref-type="bibr">43</xref>), have been
determined. Superposition of structures of MERS-CoV, SARS-CoV, and
HCoV-229E macro domains shows that the major structural divergence lies
in the α1 helices, which participates in stabilization of
ADP-ribose (<xref ref-type="fig" rid="F6">Fig.
6</xref><italic>B</italic>). Of note, in terms of the ADP-ribose
binding pockets of the three structures, the structures of ADP-ribose
appear at different degrees of curvature at the adenine moieties. In the
MERS-CoV macro domain, the side chain of Asp-20 contacting ADP-ribose
points into the cavity that holds adenine moiety. In the SARS-CoV macro
domain, the side chain position of the equivalent residue, Asp-23,
varies significantly from that of Asp-20 in the MERS-CoV macro domain.
This variation in side chain positions for Asp residues may result from
different compositions of amino acids in the α1 helix. In the
MERS-CoV macro domain, Asp-20 forms two hydrogen bonds with the N-6 atom
in a pyrimidine ring of ADP-ribose and nitrogen in the Ile-22 backbone
amide in the α1 helix via the same oxygen atom on its side chain,
thereby dragging the Asp-20 side chain into the adenine cavity. In
contrast, in the SARS-CoV macro domain, Asp-23 forms a hydrogen bond
with the N-6 atom of adenine via one of the oxygen atoms in its side
chain and with nitrogen atoms in Val-25 and Lys-26 backbone amides via
another. Hydrogen bonding with Val-25 and Lys-26 of Asp-23 in the
SARS-CoV macro domain causes a variation in side chain orientation from
that for Asp-20 in the MERS-CoV macro domain. Furthermore, in the
MERS-CoV macro domain, the lengths of hydrogen bonds formed by the
Asp-20 side chain with Ile-22 and ADP-ribose are 2.96 and 2.82 Å,
respectively. In the SARS-CoV macro domain, the lengths of hydrogen
bonds formed by the Asp-23 side chain with Val-25 and ADP-ribose are
3.04 and 2.87 Å, respectively (<xref ref-type="fig" rid="F6">Fig.
6</xref><italic>C</italic>). The differential strength of hydrogen
bonds formed by Asp with ADP-ribose and residues in the α1 helix
of the MERS-CoV and SARS-CoV macro domains may result from the presence
of different residues in α1 helices that cause variations in side
chain orientation of Asp residues in both structures. As compared with
Asp-20 in MERS-CoV and Asp-23 in SARS-CoV, the equivalent residue in
HCoV-229E is Asp-19, which does not contact ADP-ribose. Instead of
forming a hydrogen bond directly with ADP-ribose, the side chain of
Asp-19 in HCoV-229E contacts Thr-22 in the α1 helix via hydrogen
bonding with oxygen and nitrogen atoms in the side chain and backbone of
Thr-22, respectively. Hydrogen bonding with Thr-22 drags the side chain
of Asp-19 in the HCoV-229E macro domain away from the adenine cavity as
compared with the position of Asp-20 in the MERS-CoV macro domain (<xref ref-type="fig" rid="F6">Fig. 6</xref><italic>D</italic>). Consistent
with the previous study, the thermodynamic profile
(Δ<italic>G</italic> < 0, Δ<italic>H</italic> < 0,
and −<italic>T</italic>Δ<italic>S</italic> < 0) of
ADP-ribose binding to the HCoV-229E macro domain suggests less
contribution of the hydrogen bond to stabilization of ADP-ribose (<xref rid="B41" ref-type="bibr">41</xref>) (<xref rid="T2" ref-type="table">Table 2</xref>). Variations in strength of the hydrogen
bond and orientation of the side chain in Asp residues may result in
differential binding affinities of ADP-ribose observed in macro domains
of MERS-CoV (<italic>K<sub>d</sub></italic> 2.95 μ<sc>m</sc>),
SARS-CoV (<italic>K<sub>d</sub></italic> 24 μ<sc>m</sc>) (<xref rid="B36" ref-type="bibr">36</xref>), and HCoV-229E
(<italic>K<sub>d</sub></italic> 28.9 μ<sc>m</sc>) (<xref rid="B41" ref-type="bibr">41</xref>). The relationship between
binding affinities of ADP-ribose in macro domains and differential
pathogenicity of human CoVs needs further investigation.</p></sec><sec sec-type="conclusions"><title>Conclusion</title><p>Taken together, our biochemical study shows higher binding affinity for
ADP-ribose in the MERS-CoV macro domain than macro domains of CoVs
characterized to date. Structural analysis revealed that differences in
the context of hydrogen bonds formed by the conserved Asp with
ADP-ribose and residues in α1 helices in macro domains of
MERS-CoV, SARS-CoV, and HCoV-229E may result in differential binding
affinities for ADP-ribose. Our studies provide a biochemical basis for
further investigating the role of macro domain in MERS-CoV infection and
also the precise structural information for the design of novel
antiviral drugs.</p></sec></sec></sec></sec><sec><title>Author Contributions</title><p>C. H. H. conceived the study. C. C. C. and M. H. L. performed purification of the
enzyme, biochemical assays, DSF, ITC, and crystallization. C. C. C., M. H. L., and
C. Y. C. collected x-ray data. C. C. C. and C. H. H. determined and analyzed the
crystal structure. C. C. C. and C. H. H. contributed to the manuscript writing. All
authors reviewed the results and approved the final version of the manuscript.</p></sec></body><back><fn-group><fn fn-type="supported-by" id="FN1"><label>*</label><p>This work was supported by the Ministry of Science and Technology Taiwan Grant
103-2113-M-002-009-MY2 and National Taiwan University Grants NTU-ERP-104R8600
and NTU-ICRP-104R7560-5. The authors declare that they have no conflicts of
interest with the contents of this article.</p></fn><fn fn-type="other"><p>The atomic coordinates and structure factors (code <ext-link ext-link-type="uri" xlink:href="http://www.pdb.org/pdb/explore/explore.do?structureId=5DUS">5DUS</ext-link>) have been deposited in the Protein Data Bank
(<ext-link ext-link-type="uri" xlink:href="http://www.pdb.org/">http://wwpdb.org/</ext-link>).</p></fn></fn-group><fn-group content-type="abbreviations"><fn id="FN2"><label>2</label><p>The abbreviations used are: <def-list><def-item><term id="G1">SARS</term><def><p>severe acute respiratory syndrome</p></def></def-item><def-item><term id="G2">MERS</term><def><p>Middle East respiratory syndrome</p></def></def-item><def-item><term id="G3">CoV</term><def><p>coronavirus</p></def></def-item><def-item><term id="G4">NSP</term><def><p>non-structural protein</p></def></def-item><def-item><term id="G5">DSF</term><def><p>differential scanning fluorimetry</p></def></def-item><def-item><term id="G6">ITC</term><def><p>isothermal titration calorimetry</p></def></def-item><def-item><term id="G7">FCoV</term><def><p>feline CoV</p></def></def-item><def-item><term id="G8">r.m.s.</term><def><p>root mean square</p></def></def-item><def-item><term id="G9">PDB</term><def><p>Protein Data Bank.</p></def></def-item></def-list></p></fn></fn-group><ack><title>Acknowledgments</title><p>We thank the Technology Commons, College of Life Science and Center for Systems
Biology, National Taiwan University, for instrument support for protein
crystallization. Portions of this research were carried out at the National
Synchrotron Radiation Research Center, a national user facility supported by the
National Science Council of Taiwan. The Synchrotron Radiation Protein
Crystallography Facility is supported by the National Core Facility Program for
Biotechnology. We also thank Laura Smales for copyediting the manuscript.</p></ack><ref-list><title>References</title><ref id="B1"><label>1.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Hui</surname><given-names>D.
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