Structural model of the SARS coronavirus E channel in LMPG micelles
Identifieur interne : 000821 ( Pmc/Corpus ); précédent : 000820; suivant : 000822Structural model of the SARS coronavirus E channel in LMPG micelles
Auteurs : Wahyu Surya ; Yan Li ; Jaume TorresSource :
- Biochimica et Biophysica Acta. Biomembranes [ 0005-2736 ] ; 2018.
Abstract
Coronaviruses (CoV) cause common colds in humans, but are also responsible for the recent Severe Acute, and Middle East, respiratory syndromes (SARS and MERS, respectively). A promising approach for prevention are live attenuated vaccines (LAVs), some of which target the envelope (E) protein, which is a small membrane protein that forms ion channels. Unfortunately, detailed structural information is still limited for SARS-CoV E, and non-existent for other CoV E proteins. Herein, we report a structural model of a SARS-CoV E construct in LMPG micelles with, for the first time, unequivocal intermolecular NOEs. The model corresponding to the detergent-embedded region is consistent with previously obtained orientational restraints obtained in lipid bilayers and in vivo escape mutants. The C-terminal domain is mostly α-helical, and extramembrane intermolecular NOEs suggest interactions that may affect the TM channel conformation.
Url:
DOI: 10.1016/j.bbamem.2018.02.017
PubMed: 29474890
PubMed Central: 7094280
Links to Exploration step
PMC:7094280Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Structural model of the SARS coronavirus E channel in LMPG micelles</title><author><name sortKey="Surya, Wahyu" sort="Surya, Wahyu" uniqKey="Surya W" first="Wahyu" last="Surya">Wahyu Surya</name></author><author><name sortKey="Li, Yan" sort="Li, Yan" uniqKey="Li Y" first="Yan" last="Li">Yan Li</name></author><author><name sortKey="Torres, Jaume" sort="Torres, Jaume" uniqKey="Torres J" first="Jaume" last="Torres">Jaume Torres</name></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">29474890</idno><idno type="pmc">7094280</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7094280</idno><idno type="RBID">PMC:7094280</idno><idno type="doi">10.1016/j.bbamem.2018.02.017</idno><date when="2018">2018</date><idno type="wicri:Area/Pmc/Corpus">000821</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000821</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Structural model of the SARS coronavirus E channel in LMPG micelles</title><author><name sortKey="Surya, Wahyu" sort="Surya, Wahyu" uniqKey="Surya W" first="Wahyu" last="Surya">Wahyu Surya</name></author><author><name sortKey="Li, Yan" sort="Li, Yan" uniqKey="Li Y" first="Yan" last="Li">Yan Li</name></author><author><name sortKey="Torres, Jaume" sort="Torres, Jaume" uniqKey="Torres J" first="Jaume" last="Torres">Jaume Torres</name></author></analytic><series><title level="j">Biochimica et Biophysica Acta. Biomembranes</title><idno type="ISSN">0005-2736</idno><idno type="eISSN">1879-2642</idno><imprint><date when="2018">2018</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Coronaviruses (CoV) cause common colds in humans, but are also responsible for the recent Severe Acute, and Middle East, respiratory syndromes (SARS and MERS, respectively). A promising approach for prevention are live attenuated vaccines (LAVs), some of which target the envelope (E) protein, which is a small membrane protein that forms ion channels. Unfortunately, detailed structural information is still limited for SARS-CoV E, and non-existent for other CoV E proteins. Herein, we report a structural model of a SARS-CoV E construct in LMPG micelles with, for the first time, unequivocal intermolecular NOEs. The model corresponding to the detergent-embedded region is consistent with previously obtained orientational restraints obtained in lipid bilayers and in vivo escape mutants. The C-terminal domain is mostly α-helical, and extramembrane intermolecular NOEs suggest interactions that may affect the TM channel conformation.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Holmes, K V" uniqKey="Holmes K">K.V. Holmes</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Raj, V S" uniqKey="Raj V">V.S. Raj</name></author><author><name sortKey="Osterhaus, A D M E" uniqKey="Osterhaus A">A.D.M.E. Osterhaus</name></author><author><name sortKey="Fouchier, R A M" uniqKey="Fouchier R">R.A.M. Fouchier</name></author><author><name sortKey="Haagmans, B L" uniqKey="Haagmans B">B.L. Haagmans</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lou, Z" uniqKey="Lou Z">Z. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Biochim Biophys Acta Biomembr</journal-id><journal-id journal-id-type="iso-abbrev">Biochim Biophys Acta Biomembr</journal-id><journal-title-group><journal-title>Biochimica et Biophysica Acta. Biomembranes</journal-title></journal-title-group><issn pub-type="ppub">0005-2736</issn><issn pub-type="epub">1879-2642</issn><publisher><publisher-name>Elsevier</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">29474890</article-id><article-id pub-id-type="pmc">7094280</article-id><article-id pub-id-type="publisher-id">S0005-2736(18)30058-0</article-id><article-id pub-id-type="doi">10.1016/j.bbamem.2018.02.017</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Structural model of the SARS coronavirus E channel in LMPG micelles</article-title></title-group><contrib-group><contrib contrib-type="author" id="au0005"><name><surname>Surya</surname><given-names>Wahyu</given-names></name></contrib><contrib contrib-type="author" id="au0010"><name><surname>Li</surname><given-names>Yan</given-names></name></contrib><contrib contrib-type="author" id="au0015"><name><surname>Torres</surname><given-names>Jaume</given-names></name><email>jtorres@ntu.edu.sg</email><xref rid="cr0005" ref-type="corresp">⁎</xref></contrib></contrib-group><aff id="af0005">School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore</aff><author-notes><corresp id="cr0005"><label>⁎</label>Corresponding author. <email>jtorres@ntu.edu.sg</email></corresp></author-notes><pub-date pub-type="pmc-release"><day>21</day><month>2</month><year>2018</year></pub-date><pmc-comment> PMC Release delay is 0 months and 0 days and was based on .</pmc-comment>
<pub-date pub-type="ppub"><month>6</month><year>2018</year></pub-date><pub-date pub-type="epub"><day>21</day><month>2</month><year>2018</year></pub-date><volume>1860</volume><issue>6</issue><fpage>1309</fpage><lpage>1317</lpage><history><date date-type="received"><day>1</day><month>11</month><year>2017</year></date><date date-type="rev-recd"><day>14</day><month>2</month><year>2018</year></date><date date-type="accepted"><day>16</day><month>2</month><year>2018</year></date></history><permissions><copyright-statement>© 2018 Elsevier B.V.</copyright-statement><copyright-year>2018</copyright-year><copyright-holder>Elsevier B.V.</copyright-holder><license><license-p>Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.</license-p></license></permissions><abstract id="ab0005"><p>Coronaviruses (CoV) cause common colds in humans, but are also responsible for the recent Severe Acute, and Middle East, respiratory syndromes (SARS and MERS, respectively). A promising approach for prevention are live attenuated vaccines (LAVs), some of which target the envelope (E) protein, which is a small membrane protein that forms ion channels. Unfortunately, detailed structural information is still limited for SARS-CoV E, and non-existent for other CoV E proteins. Herein, we report a structural model of a SARS-CoV E construct in LMPG micelles with, for the first time, unequivocal intermolecular NOEs. The model corresponding to the detergent-embedded region is consistent with previously obtained orientational restraints obtained in lipid bilayers and in vivo escape mutants. The C-terminal domain is mostly α-helical, and extramembrane intermolecular NOEs suggest interactions that may affect the TM channel conformation.</p></abstract><abstract abstract-type="graphical" id="ab0010"><title>Graphical abstract</title><p><fig id="f0045" position="anchor"><alt-text id="al0045">Unlabelled Image</alt-text><graphic xlink:href="fx1_lrg"></graphic></fig></p></abstract><abstract abstract-type="author-highlights" id="ab0015"><title>Highlights</title><p><list list-type="simple" id="l0005"><list-item id="li0005"><label>•</label><p id="p0005">SARS-CoV E protein is a pentameric ion channel.</p></list-item><list-item id="li0010"><label>•</label><p id="p0010">The SARS-CoV E protein (8–65) is almost completely α-helical in LMPG micelles.</p></list-item><list-item id="li0015"><label>•</label><p id="p0015">Ten inter-monomeric NOEs have been identified.</p></list-item><list-item id="li0020"><label>•</label><p id="p0020">The SARS-CoV E protein (8–65) pentameric model has been obtained.</p></list-item><list-item id="li0025"><label>•</label><p id="p0025">Orientation of key residues, e.g. Val25 is consistent with previous in vivo results.</p></list-item></list></p></abstract><kwd-group id="ks0005"><title>Abbreviations</title><kwd>CoV, coronavirus</kwd><kwd>SARS, severe acute respiratory syndrome</kwd><kwd>MERS, Middle East respiratory syndrome</kwd><kwd>E, envelope</kwd><kwd>M, membrane</kwd><kwd>TM, transmembrane</kwd><kwd>PBM, PDZ-binding motif</kwd><kwd>IC, ion channel</kwd><kwd>LAV, live attenuated vaccine</kwd><kwd>DPC, n-dodecyl-phosphocholine</kwd><kwd>LMPG, lyso-myristoyl phosphatidylglycerol</kwd><kwd>PFO, perfluoro octanoic acid</kwd><kwd>DMPC, dimyristoyl phosphatidylcholine</kwd><kwd>HMA, hexamethylene amiloride</kwd><kwd>BN-PAGE, blue-native polyacrylamide gel electrophoresis</kwd><kwd>AQPZ, aquaporin Z</kwd><kwd>CSP, chemical shift perturbation</kwd></kwd-group><kwd-group id="ks0010"><title>Keywords</title><kwd>Envelope protein</kwd><kwd>Solution NMR</kwd><kwd>Transmembrane α-helices</kwd><kwd>Micelles</kwd><kwd>Oligomerization</kwd></kwd-group></article-meta></front><body><sec id="s0005"><label>1</label><title>Introduction</title><p id="p0030">Coronaviruses (CoV) typically affect the respiratory tract and gut of mammals and birds. Approximately 30% of common colds are caused by two human coronaviruses - OC43 and 229E. Of particular interest are the viruses responsible for the severe acute respiratory syndrome (SARS), which produced a near pandemic in 2003 [<xref rid="bb0005" ref-type="bibr">1</xref>], and the recent Middle East respiratory syndrome coronavirus (MERS-CoV) [<xref rid="bb0010" ref-type="bibr">2</xref>].</p><p id="p0035">No effective licensed treatments exist against coronavirus infections [<xref rid="bb0015" ref-type="bibr">[3]</xref>, <xref rid="bb0020" ref-type="bibr">[4]</xref>, <xref rid="bb0025" ref-type="bibr">[5]</xref>], but live attenuated vaccines (LAVs) [<xref rid="bb0030" ref-type="bibr">[6]</xref>, <xref rid="bb0035" ref-type="bibr">[7]</xref>, <xref rid="bb0040" ref-type="bibr">[8]</xref>, <xref rid="bb0045" ref-type="bibr">[9]</xref>, <xref rid="bb0050" ref-type="bibr">[10]</xref>] and fusion inhibitors [<xref rid="bb0055" ref-type="bibr">11</xref>] are promising strategies. One CoV component critical for pathogenesis is the envelope (E) protein, as reported in several coronaviruses, e.g., MERS and SARS-CoVs [<xref rid="bb0060" ref-type="bibr">[12]</xref>, <xref rid="bb0065" ref-type="bibr">[13]</xref>, <xref rid="bb0070" ref-type="bibr">[14]</xref>]. The CoV envelope (E) proteins are short polypeptides (76–109 amino acids) with a single α-helical transmembrane (TM) domain [<xref rid="bb0075" ref-type="bibr">[15]</xref>, <xref rid="bb0080" ref-type="bibr">[16]</xref>, <xref rid="bb0085" ref-type="bibr">[17]</xref>, <xref rid="bb0090" ref-type="bibr">[18]</xref>, <xref rid="bb0095" ref-type="bibr">[19]</xref>, <xref rid="bb0100" ref-type="bibr">[20]</xref>, <xref rid="bb0105" ref-type="bibr">[21]</xref>] that form homopentameric ion channels (IC) with poor ion selectivity [<xref rid="bb0110" ref-type="bibr">22</xref>,<xref rid="bb0115" ref-type="bibr">23</xref>]. CoV E proteins are mostly found in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) [<xref rid="bb0120" ref-type="bibr">[24]</xref>, <xref rid="bb0125" ref-type="bibr">[25]</xref>, <xref rid="bb0130" ref-type="bibr">[26]</xref>, <xref rid="bb0135" ref-type="bibr">[27]</xref>, <xref rid="bb0140" ref-type="bibr">[28]</xref>, <xref rid="bb0145" ref-type="bibr">[29]</xref>]. In animal models, deletion of SARS-CoV E protein reduced pathogenicity and mortality [<xref rid="bb0150" ref-type="bibr">30</xref>], whereas cellular models displayed up- and down-regulation of stress response and inflammation host genes, respectively [<xref rid="bb0155" ref-type="bibr">31</xref>]. The importance of E protein in pathogenesis has led to the development of LAVs based on deletion of E protein in SARS- and MERS-CoVs, although this led to compensatory mechanisms that recover virulence [<xref rid="bb0160" ref-type="bibr">32</xref>,<xref rid="bb0165" ref-type="bibr">33</xref>].</p><p id="p0040">Specific critical features in the SARS-CoV E protein sequence have been identified that determine virulence, e.g., at the C-terminal tail [<xref rid="bb0170" ref-type="bibr">34</xref>] or in the TM domain [<xref rid="bb0150" ref-type="bibr">30</xref>], and precise structural characterization of these regions could help in the design of E protein-based CoV LAVs. However, detailed structural knowledge is still very limited in the case of SARS-CoV E, and non-existent for other CoV E proteins.</p><p id="p0045">A pentameric model for SARS-CoV E was initially proposed by the authors after an in silico conformational search [<xref rid="bb0075" ref-type="bibr">15</xref>] of TM domain oligomers. In that report, two pentameric models (termed ‘A’ and ‘B’) that were separated by a ~50° rotation of their α-helices were selected. In model A, V25 adopts a more lumenal position, whereas in model B, the position of this residue is clearly interhelical (<xref rid="f0005" ref-type="fig">Fig. 1</xref>). The pentameric organization of SARS-CoV E has been confirmed experimentally in various detergents: PFO, DPC or C-14 betaine [<xref rid="bb0085" ref-type="bibr">17</xref>,<xref rid="bb0090" ref-type="bibr">18</xref>], not only for synthetic TM (E<sub>TM</sub>), but also for an 8–65 (E<sub>TR</sub>) construct and for full length E protein (E<sub>FL</sub>).<fig id="f0005"><label>Fig. 1</label><caption><p>Comparison of orientation of residue V25 in SARS-CoV E<sub>TM</sub> pentameric models. Orientation of computational models A (orange) and B (cyan) [<xref rid="bb0075" ref-type="bibr">15</xref>], where the side chain of V25 (F26 is only used to guide the eye) is indicated. The ‘A-like’ model obtained by NMR [<xref rid="bb0100" ref-type="bibr">20</xref>] is shown in red. In model B, the position of V25 is clearly interhelical.</p></caption><alt-text id="al0005">Fig. 1</alt-text><graphic xlink:href="gr1_lrg"></graphic></fig></p><p id="p0050">To confirm experimentally the orientation of the α-helices in the pentameric model, site specific infrared dichroism (SSID) measurements [<xref rid="bb0175" ref-type="bibr">35</xref>] were obtained in hydrated lipid bilayers, with <sup>13</sup>C = <sup>18</sup>O isotopically labeled synthetic E<sub>TM</sub>. However, the orientation of the α-helices turned out to be strongly dependent on the presence of 2 flanking lysine residues at each end of the peptides [<xref rid="bb0080" ref-type="bibr">16</xref>]: with flanking lysine residues, the orientation was a hybrid between models A and B (residues 17–24 were oriented consistent with model B, but from residue 24 onwards, orientation was as expected for model A), consistent with a ‘bend of the α-helices around residues 25–27’ [<xref rid="bb0080" ref-type="bibr">16</xref>]. Without terminal lysines, however, the orientation of the central five labeled consecutive residues, L21 to V25, was entirely consistent with model A [<xref rid="bb0085" ref-type="bibr">17</xref>].</p><p id="p0055">These initial results suggested that the conformation of the E<sub>TM</sub> pentamer may be very sensitive of the presence of extra residues and probably also, extramembrane domains. An NMR study was performed on a synthetic E<sub>TM</sub> (residues 8–38) in DPC detergent micelles, where E<sub>TM</sub> was selectively labeled [<xref rid="bb0100" ref-type="bibr">20</xref>]. E<sub>TM</sub> was <sup>15</sup>N-labeled at A22, V24, V25, and <sup>13</sup>C, <sup>15</sup>N-labeled at L18, L19 and L21. Intermonomeric NOEs were assigned indirectly, i.e., when cross-peaks could not be explained by intramonomer interactions. Of these, derived from difference 2D homonuclear <sup>1</sup>H<sup>N</sup>, <sup>1</sup>H<sup>aromatic</sup> band-selected NOESY, only four NOEs were labeled ‘strong’, and involved the <sup>1</sup>H<sup>δε</sup> phenyl ring of Phe23, to <sup>1</sup>H<sub>3</sub><sup>δ1</sup>/<sup>1</sup>H<sub>3</sub><sup>δ2</sup> of either Leu18 (two NOEs) or Leu21 (two NOEs). These intermolecular NOEs were insufficient to distinguish between models A and B, and the monomer structure was fit to a model A template.</p><p id="p0060">More recently, recombinant SARS-CoV escape mutants were recovered after introducing a V25F channel-inactivating mutation in the E protein, [<xref rid="bb0180" ref-type="bibr">36</xref>], that led to attenuation in a mouse model [<xref rid="bb0150" ref-type="bibr">30</xref>]. Revertant mutants regained fitness and pathogenicity whereas mutated E protein regained channel activity [<xref rid="bb0150" ref-type="bibr">30</xref>]. Surprisingly, escape mutations in E protein clustered along the helix <italic>interface</italic> opposite to residue V25, consistent with an interhelical orientation of this residue, as found in model B (<xref rid="f0005" ref-type="fig">Fig. 1</xref>, cyan).</p><p id="p0065">In the present paper, we report a more accurate model of the SARS-CoV E protein pentamer, in LMPG micelles. The construct we have used prolongs the TM domain with another 27 residues in the C-terminal domain (residues 8–65). Following established protocols [<xref rid="bb0185" ref-type="bibr">37</xref>], two types of monomers were mixed, bearing different isotopical labels, that allowed unambiguous identification of ten intermonomeric NOEs. In a nutshell, the results are consistent with a TM model that appears to be a hybrid between models A and B: while overall being closer to model A, residue V25 has a clear ‘model B-like’ interhelical orientation, consistent with the revertant mutants that appeared <italic>in vivo</italic>.</p></sec><sec id="s0010"><label>2</label><title>Materials and methods</title><sec id="s0015"><label>2.1</label><title>Protein expression and purification</title><p id="p0070">The expression and purification methods for the truncated SARS-CoV E construct corresponding to residues 8–65 (E<sub>TR</sub>) have been described previously [<xref rid="bb0095" ref-type="bibr">19</xref>]. This construct does not have cysteines, as these are not required for oligomerization [<xref rid="bb0090" ref-type="bibr">18</xref>,<xref rid="bb0095" ref-type="bibr">19</xref>,<xref rid="bb0140" ref-type="bibr">28</xref>,<xref rid="bb0190" ref-type="bibr">38</xref>]. In the present work, M9 media was supplemented with an appropriate combination of <sup>15</sup>NH<sub>4</sub>Cl, <sup>13</sup>C-glucose, <sup>2</sup>H-glucose, and <sup>2</sup>H<sub>2</sub>O (Cambridge Isotope Laboratories) to produce<sup>15</sup>N-, <sup>13</sup>C-, <sup>15</sup>N/<sup>13</sup>C- and <sup>15</sup>N/<sup>2</sup>H-labeled E<sub>TR</sub> samples. For preparation of fully deuterated <sup>15</sup>N/<sup>2</sup>H-labeled samples, freshly transformed <italic>E. coli</italic> cells were doubly-selected in LB agar plates and media prepared with 30% and 60% <sup>2</sup>H<sub>2</sub>O, successively, and later grown in M9 media prepared with 99.9% <sup>2</sup>H<sub>2</sub>O [<xref rid="bb0195" ref-type="bibr">39</xref>,<xref rid="bb0200" ref-type="bibr">40</xref>].</p></sec><sec id="s0020"><label>2.2</label><title>Gel electrophoresis</title><p id="p0075">Blue-native PAGE (BN-PAGE) was performed as described previously [<xref rid="bb0205" ref-type="bibr">41</xref>]. Lyophilized E<sub>TR</sub> protein was solubilized (0.1 mM) in sample buffer containing LMPG (lyso-myristoyl phosphatidylglycerol, Anatrace) at the indicated concentrations.</p></sec><sec id="s0025"><label>2.3</label><title>Residue rotational pitch calculations</title><p id="p0080">For α-helical bundle models, the rotational pitch angle of a residue, ω, defined arbitrarily as 0° or 180° when transition dipole moment, helix director, and the z-axis all reside in a single plane, was calculated as described elsewhere [<xref rid="bb0210" ref-type="bibr">42</xref>]. The final result is the average of the ω values calculated in each monomer. For a canonical α-helix, it is expected that Δω between two consecutive residues is ~100°.</p></sec><sec id="s0030"><label>2.4</label><title>NMR sample preparation</title><p id="p0085">Lyophilized E<sub>TR</sub> protein (0.67 mM) was solubilized in 20 mM sodium phosphate pH 5.5, 50 mM NaCl, and 200 mM LMPG, i.e., a protein:detergent (P/D) molar ratio of 1:300. The same protein concentration and P/D ratio was used for the mixture of <sup>15</sup>N-D and <sup>13</sup>C-labeled samples. The solution was vortexed and sonicated several times until a clear solution was obtained, indicating protein reconstitution into detergent micelles.</p></sec><sec id="s0035"><label>2.5</label><title>NMR spectroscopy</title><p id="p0090">NMR experiments were performed at 308 K using an Avance-II 700 NMR spectrometer with cryogenic probes. Sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) was used as the internal reference for <sup>1</sup>H nuclei. The chemical shifts of <sup>13</sup>C and <sup>15</sup>N nuclei were calculated from the <sup>1</sup>H chemical shifts. The NMR data were processed using TopSpin 3.1 (<ext-link ext-link-type="uri" xlink:href="http://www.bruker-biospin.com" id="ir0005">www.bruker-biospin.com</ext-link>) and analyzed using CARA (<ext-link ext-link-type="uri" xlink:href="http://www.nmr.ch" id="ir0010">www.nmr.ch</ext-link>). Sequence-specific assignment of backbone <sup>1</sup>H<sup>N</sup>, <sup>15</sup>N, <sup>13</sup>C′ and <sup>13</sup>C<sup>α</sup> was achieved by using 2D [<sup>1</sup>H-<sup>15</sup>N]-TROSY-HSQC, 3D HNCA and HN(CO)CA experiments on a <sup>15</sup>N/<sup>13</sup>C-labeled E<sub>TR</sub> protein. Side-chain resonances were assigned using 3D <sup>15</sup>N-resolved NOESY-HSQC (120 ms mixing time), (H)CCH-TOCSY and <sup>13</sup>C-resolved NOESY-HSQC (120 ms mixing time). To identify membrane-embedded residues, the NMR sample was lyophilized overnight and reconstituted in 99% D<sub>2</sub>O. Immediately after reconstitution, 2D [<sup>1</sup>H-<sup>15</sup>N]-TROSY was collected. The titration experiments with 5-(<italic>N</italic>,<italic>N</italic>-hexamethylene) amiloride (HMA) were performed with <sup>15</sup>N-labeled E<sub>TR</sub> sample. Chemical shift perturbation (CSP) values and chemical shift differences were calculated using the formula CSP <inline-formula><mml:math id="M1" altimg="si1.gif" overflow="scroll"><mml:mo>=</mml:mo><mml:msqrt><mml:mrow><mml:mo>△</mml:mo><mml:msup><mml:mi>δH</mml:mi><mml:mn>2</mml:mn></mml:msup><mml:mo>+</mml:mo><mml:msup><mml:mfenced open="(" close=")"><mml:mrow><mml:mn>0.23</mml:mn><mml:mo>∗</mml:mo><mml:mo>△</mml:mo><mml:mi>δN</mml:mi></mml:mrow></mml:mfenced><mml:mn>2</mml:mn></mml:msup></mml:mrow></mml:msqrt></mml:math></inline-formula>.</p></sec><sec id="s0040"><label>2.6</label><title>Structure calculation</title><p id="p0095">Intra-monomeric NOE distance restraints were obtained from <sup>15</sup>N-NOESY-HSQC and <sup>13</sup>C-NOESY-HSQC spectra (both with a mixing time of 120 ms). Backbone dihedral angle restraints (φ and ψ) were derived from <sup>13</sup>C′, <sup>13</sup>C<sup>α</sup>, <sup>13</sup>C<sup>β</sup>, <sup>1</sup>H<sup>α</sup> and <sup>1</sup>H<sup>β</sup> chemical shift values using TALOS+ [<xref rid="bb0215" ref-type="bibr">43</xref>]. Short-range and medium range NOE connectivities were used to establish sequence-specific <sup>1</sup>H NMR assignments and to identify elements of the regular secondary structure. Hydrogen bonds were derived from the NOE connectivity, and supported by the H/D exchange data. Monomer structure calculations were performed using CYANA 3.0 [<xref rid="bb0220" ref-type="bibr">44</xref>,<xref rid="bb0225" ref-type="bibr">45</xref>] and visualized using PyMOL (Delano Scientific). All of the restraints used in the calculations to obtain a total of 10 monomer structures, and all the structure statistics, are summarized in Supplementary Tables S1 and S2.</p><p id="p0100">Inter-monomeric NOE restraints were obtained from 3D <sup>15</sup>N-resolved NOESY-HSQC (250 ms mixing time) of two sets of asymmetrically deuterated samples: (1) <sup>15</sup>N/<sup>2</sup>H-labeled E<sub>TR</sub> sample (ND), and (2) an equimolar mixture of <sup>15</sup>N/<sup>2</sup>H-labeled and a non-deuterated <sup>13</sup>C-labeled E<sub>TR</sub> sample (ND + C). NOE cross-peaks appearing in sample ND + C but not in sample ND were assigned to inter-monomeric contacts. Conversely, resonances also appearing in the ND sample were attributed to incomplete deuteration, and were assigned to intra-monomeric NOEs.</p><p id="p0105">The pentamer structure was calculated using HADDOCK 2.2 [<xref rid="bb0230" ref-type="bibr">46</xref>] according to standard protocols. Ten inter-monomeric NOE restraints (defined as above) were described as ambiguous and unambiguous 5.0 Å distance restraints. Two segments were described as fully flexible: residues 37–47 and 40–54. A C5 symmetry restraint between all 5 subunits and pairwise non-crystallographic symmetry restraints between neighbouring subunits were applied. Initial rigid-body docking yielded 1000 structures, out of which 200 top-scoring structures (i.e., based on HADDOCK target function score) were selected for refinement by semi-flexible simulated annealing. These were then clustered based on RMSD, and the top-scoring cluster was selected (all 16 structures within the said cluster were grouped to form an ensemble).</p></sec></sec><sec id="s0045"><label>3</label><title>Results and discussion</title><sec id="s0050"><label>3.1</label><title>Helical structure and TM domain of SARS-CoV E monomer (E<sub>TR</sub>) in LMPG micelles</title><p id="p0110">Despite phospholipid isotropic bicelles may have been more membrane-like than detergent micelles, in our hands, phospholipid bicelles did not produce suitable spectra of E<sub>TR</sub> (not shown). Examples of significant differences observed in bicelles vs micelles have been reported, e.g., in the study of the integrin TM heterodimers [<xref rid="bb0325" ref-type="bibr">[47]</xref>, <xref rid="bb0330" ref-type="bibr">[48]</xref>, <xref rid="bb0335" ref-type="bibr">[49]</xref>, <xref rid="bb0340" ref-type="bibr">[50]</xref>, <xref rid="bb0345" ref-type="bibr">[51]</xref>, <xref rid="bb0350" ref-type="bibr">[52]</xref>] or in viral channels [<xref rid="bb0355" ref-type="bibr">53</xref>].</p><p id="p0115">Nevertheless, we have shown previously that E<sub>TR</sub> is pentameric in various detergents [<xref rid="bb0085" ref-type="bibr">17</xref>,<xref rid="bb0090" ref-type="bibr">18</xref>], although none of them was suitable for NMR studies of E<sub>TR</sub> or E<sub>FL</sub> (not shown). E<sub>TR</sub> only produced reasonably good NMR spectra in DPC when SDS was also present [<xref rid="bb0095" ref-type="bibr">19</xref>], but since SDS disrupts E<sub>TR</sub> oligomerization, we searched for other micellar environments. Lipid-like LMPG was found to produce good NMR spectra for E<sub>TR</sub>, although not for E<sub>FL</sub>. Therefore, E<sub>TR</sub> in LMPG was used in subsequent experiments. The use of the E<sub>TR</sub> construct instead of the full-length E protein (E<sub>FL</sub>) is justified since the <sup>13</sup>Cα chemical shifts of E<sub>TR</sub> and E<sub>FL</sub> protein in SDS or SDS/DPC were almost identical for residues 8–65 [<xref rid="bb0095" ref-type="bibr">19</xref>]. In addition, the secondary structure, obtained by CD/FTIR [<xref rid="bb0090" ref-type="bibr">18</xref>], of E<sub>TR</sub> and E<sub>FL</sub> is similar and predominantly α-helical, whether in DPC, SDS, mixed (1:2 M ratio) SDS/DPC micelles or DMPC synthetic membranes [<xref rid="bb0090" ref-type="bibr">18</xref>,<xref rid="bb0095" ref-type="bibr">19</xref>].</p><p id="p0120">Comparison of the HSQC spectrum of E<sub>TR</sub>/LMPG before and after exposure to D<sub>2</sub>O (<xref rid="f0010" ref-type="fig">Fig. 2</xref>A) shows that only 20 residues are protected from hydrogen/deuterium (H/D) exchange. The protected residues correspond to the stretch L18-L37, unequivocally indicating the presence of a <italic>single</italic> TM domain in SARS-CoV E. This result is consistent with the stretch L18-L39 found to be protected in SDS micelles [<xref rid="bb0095" ref-type="bibr">19</xref>]. The chemical shift index (CSI)-based secondary structure of E<sub>TR</sub> (calculated by using TALOS+) obtained in LMPG (<xref rid="f0010" ref-type="fig">Fig. 2</xref>B), has significantly higher helicity in C-terminal residues 52–55, when compared with the data obtained SDS or with a mixture SDS/DPC [<xref rid="bb0095" ref-type="bibr">19</xref>].<fig id="f0010"><label>Fig. 2</label><caption><p>Hydrogen-deuterium exchange protected region and secondary structure of E<sub>TR</sub> monomer in LMPG. (A) [<sup>1</sup>H-<sup>15</sup>N]-TROSY-HSQC spectra in H<sub>2</sub>O (left) and 99% D<sub>2</sub>O (right), with cross-peaks labeled by one-letter code and residue number; (B) Secondary structure prediction obtained using TALOS+ [<xref rid="bb0215" ref-type="bibr">43</xref>], comparing E<sub>TR</sub> in LMPG, SDS, and SDS/DPC [<xref rid="bb0095" ref-type="bibr">19</xref>]. (Layout note: 1 column).</p></caption><alt-text id="al0010">Fig. 2</alt-text><graphic xlink:href="gr2_lrg"></graphic></fig></p><p id="p0125">The structure of E<sub>TR</sub> was calculated from 10 E<sub>TR</sub> monomer structures (<xref rid="f0015" ref-type="fig">Fig. 3</xref>A) and the structure statistics are summarized in Supplementary Table S1. The E<sub>TR</sub> monomer in LMPG consists of three helical segments: the one encompassing the TM domain (H1, residues 12–37), a juxtamembrane middle helical segment (H2, residues 39–47), and a C-terminal helix (H3, residues 52–65) (<xref rid="f0015" ref-type="fig">Fig. 3</xref>B). In contrast, E<sub>TR</sub> in DPC/SDS [<xref rid="bb0095" ref-type="bibr">19</xref>] was formed by only two helical segments separated by a long flexible link (<xref rid="f0015" ref-type="fig">Fig. 3</xref>C). Compared to the results in SDS or SDS/DPC [<xref rid="bb0095" ref-type="bibr">19</xref>], in LMPG helix H3 is extended by 3 residues on its N-terminal side, whereas a new helical segment, H2, is formed.<fig id="f0015"><label>Fig. 3</label><caption><p>E<sub>TR</sub> consists of three α-helical segments in LMPG. (A) Ensemble of 10 calculated E<sub>TR</sub> monomer structures in LMPG showing the backbone as line representation; (B) for clarity, the helical segments shown in (A) are superimposed locally and the side chains are shown as line representation; (C) graphical comparison of α-helical stretches and H/D protection (showing the TM domain) in LMPG obtained herein and in SDS/DPC environments [<xref rid="bb0095" ref-type="bibr">19</xref>]. Structure statistics in LMPG are summarized in Supplementary Table S1. (Layout note: 1.5 columns).</p></caption><alt-text id="al0015">Fig. 3</alt-text><graphic xlink:href="gr3_lrg"></graphic></fig></p></sec><sec id="s0055"><label>3.2</label><title>Oligomeric state of SARS-CoV E in LMPG</title><p id="p0130">To assess the oligomerization of E<sub>TR</sub> in LMPG micelles, its migration in a BN-PAGE gel was analyzed at various protein-to-detergent (P/D) ratios (<xref rid="f0020" ref-type="fig">Fig. 4</xref>). At the lowest P/D molar ratio (1:1000), E<sub>TR</sub> migrates as a ladder of increasingly larger oligomers where the fastest migrating band is assumed to correspond to monomers (lower star), ~8 kDa, whereas at a high P/D ratio (1:125), E<sub>TR</sub> migrates with an apparent molecular weight of ~150 kDa. These results are almost identical to those obtained previously for MERS-CoV E, for which a pentameric oligomer was determined using analytical ultracentrifugation in C-14 betaine. In that case, migration in BN-PAGE gels was also observed as a single ~150 kDa band in detergents DPC, DHPC and LMPG [<xref rid="bb0105" ref-type="bibr">21</xref>], and the ladder observed at higher detergent concentration conveniently provided an internal reference that served as a oligomeric size marker. Similar to E<sub>TR</sub>, by comparison with that ladder, we confidently assigned the single band observed for MERS-CoV E to pentameric oligomers. It should be noted that in BN-PAGE gels of membrane proteins, molecular weights can appear up to 80% higher due to a contribution of the dye [<xref rid="bb0360" ref-type="bibr">54</xref>]. We have shown this for tetrameric AQPZ, which migrated at ~170 kDa instead of the expected ~100 kDa, and with a viroporin, the SH protein pentamer [<xref rid="bb0205" ref-type="bibr">41</xref>], which migrated as ~66 kDa instead of ~40 kDa. In the case of envelope E proteins, the effect is even more pronounced. In both SARS-CoV E<sub>TR</sub> and MERS-CoV E, the pentameric form appears at ~150 kDa, therefore the monomer should appear at >30 kDa. This is consistent with its migration above the AQPZ monomer (~25 kDa). The ladder ends with a pentamer, which is the predominant band at high P/D ratios. The proportion of large oligomers naturally decrease at low P/D ratios, but a significant amount of pentamer species is still present even at the 1:1000 P/D ratio. The NMR data was collected at a P/D molar ratio of 1:300, which should mostly be formed by pentamers.<fig id="f0020"><label>Fig. 4</label><caption><p>Oligomeric state of SARS-CoV E in LMPG. BN-PAGE of E<sub>TR</sub> in lipid-like LMPG detergent (peptide-to-detergent ratio is indicated). A ladder of oligomeric sizes is indicated by stars (*). The membrane protein aquaporin Z from <italic>E. coli</italic> (AqpZ) is used as reference, in monomeric and tetrameric forms (AqpZ:1 and AQPZ:4, respectively). (Layout note: 1 column).</p></caption><alt-text id="al0020">Fig. 4</alt-text><graphic xlink:href="gr4_lrg"></graphic></fig></p></sec><sec id="s0060"><label>3.3</label><title>HMA binding to oligomeric E<sub>TR</sub></title><p id="p0135">In a previous paper, we showed that monomeric E<sub>TR</sub> in SDS micelles was not affected by addition of the drug HMA [<xref rid="bb0095" ref-type="bibr">19</xref>]. However, after addition of DPC to SDS, to a SDS/DPC 1:4 M ratio, HMA induced clear chemical shift perturbations (CSPs), concomitant with E<sub>TR</sub> oligomerization. The oligomerization in DPC/SDS was not homogeneous, which precluded a more detailed study, whereas in LMPG a predominant oligomeric size is observed at a high protein-detergent ratio (<xref rid="f0020" ref-type="fig">Fig. 4</xref>). Therefore, in LMPG the changes observed after HMA addition should more reliably represent the binding of HMA to E<sub>TR</sub>. HMA-induced CSPs were detected herein after addition of 7.75 mM HMA to 0.25 mM E<sub>TR</sub> in 200 mM LMPG micelles (P/D molar ratio 1:800) (<xref rid="f0025" ref-type="fig">Fig. 5</xref>).<fig id="f0025"><label>Fig. 5</label><caption><p>E<sub>TR</sub> oligomer chemical shifts perturbation (CSP) by HMA. (A) Superposition of TROSY-HSQC spectra of uniformly <sup>15</sup>N-labeled E<sub>TR</sub> protein (0.25 mM monomer concentration) in the absence (red) and presence (blue) of 7.75 mM HMA. Peaks that undergo significant shifts upon HMA addition are highlighted; (B) selected regions in the TROSY-HSQC spectrum at varying HMA concentration: 0 (red), 0.25 (pink), 0.75 (purple), 1.75 (yellow), 3.75 (light green), 7.75 (green), 9.75 (light blue), 11.75 mM HMA (blue); (C) chemical shift perturbation (CSP) of the backbone amide resonances of 0.25 mM <sup>15</sup>N-labeled E<sub>TR</sub> protein upon titration with 7.75 mM HMA. Mean CSP value across all residues and the standard deviation are shown by dashed and dotted line, respectively. Missing/overlapping residues are omitted. (Layout note: 1 column).</p></caption><alt-text id="al0025">Fig. 5</alt-text><graphic xlink:href="gr5_lrg"></graphic></fig></p><p id="p0140">The average CSP value was 0.019 ppm, and several residues showed CSP >1 S.D. from the average value, notably Thr-9, Leu-12, Ile-13, Ala-36 and Val-47. These results suggest the presence of two binding sites located at both ends of the TM domain. Given the long distance between Ala-36 and Val-47, the two HMA-interacting residues may be located in different monomers.</p></sec><sec id="s0065"><label>3.4</label><title>Pentameric model of E<sub>TR</sub></title><p id="p0145">A pentameric model was obtained by docking the monomeric form of E<sub>TR</sub> using HADDOCK 2.2 [<xref rid="bb0230" ref-type="bibr">46</xref>], which incorporated 10 inter-monomeric NOE restraints (<xref rid="f0030" ref-type="fig">Fig. 6</xref>A). We note that 2 inter-monomeric NOEs are located at the extramembrane C-terminal tail: L39 HN - Y57 HB and V47 HN - N64 HN. The same figure shows a representative example of NOE E<sub>TR</sub> inter-monomer connectivity (<xref rid="f0030" ref-type="fig">Fig. 6</xref>B). The remaining plots of inter-monomeric NOEs are shown in Fig. S1. Structure statistics are summarized in Supplementary Table S2.<fig id="f0030"><label>Fig. 6</label><caption><p>Inter-monomeric NOEs in E<sub>TR</sub> pentamer. (A) List of inter-monomeric NOE contacts, with those located in the extramembrane C-terminal region in bold; (B) a representative example of NOE E<sub>TR</sub> inter-monomer connectivity (green lines). Selected strips correspond to a <sup>15</sup>N–NOESY-HSQC spectrum and NH protons of V14 for samples <sup>15</sup>N/<sup>2</sup>H-labeled (ND), <sup>15</sup>N/<sup>2</sup>H-labeled + <sup>13</sup>C-labeled (ND + C), and <sup>15</sup>N/<sup>13</sup>C-labeled (NC). The NOE strips from the NC sample are shown as reference, as they contain both intra and inter-monomer contacts. Strips corresponding to the remaining NOE connectivity are shown in Fig. S1. (Layout note: 1 column).</p></caption><alt-text id="al0030">Fig. 6</alt-text><graphic xlink:href="gr6_lrg"></graphic></fig></p><p id="p0150">The E<sub>TR</sub> pentamer is a right handed α-helical bundle where the C-terminal tails coil around each other (<xref rid="f0035" ref-type="fig">Fig. 7</xref>A) likely owing to the 2 inter-monomeric restraints between the two C-terminal helices. Each pentamer subunit (<xref rid="f0035" ref-type="fig">Fig. 7</xref>B) has better defined structure compared to the monomer alone (<xref rid="f0015" ref-type="fig">Fig. 3</xref>A). This is mainly due to decreased flexibility at the inter-helical segments, which were kept flexible during the docking, as the two C-terminal helices now adopt a relatively fixed conformation. This is also apparent from the RMSD values; the pentamer subunit RMSD values are significantly reduced as compared to the monomer (<xref rid="f0035" ref-type="fig">Fig. 7</xref>C).<fig id="f0035"><label>Fig. 7</label><caption><p>Structure of the E<sub>TR</sub> pentamer. (A) Top view of the E<sub>TR</sub> pentamer showing an ensemble of 16 structures obtained using HADDOCK and 10 inter-monomeric NOE restraints (see Materials and Methods); (B) Side view of one subunit of the pentamer showing the backbone as line representation; (C) RMSD values (per-residue) of the monomer ensemble (see <xref rid="f0015" ref-type="fig">Fig. 3</xref>A, black), structured helical segments of the monomer (see <xref rid="f0015" ref-type="fig">Fig. 3</xref>B, blue), and the pentamer ensemble (<xref rid="f0035" ref-type="fig">Fig. 7</xref>A, red). The average RMSD value of the monomer (dashed line) and ±1 S.D. values (grey band) are indicated. (Layout note: 1.5 column).</p></caption><alt-text id="al0035">Fig. 7</alt-text><graphic xlink:href="gr7_lrg"></graphic></fig></p><p id="p0155">Notably, in this pentameric model, the location of V25 is interhelical (<xref rid="f0040" ref-type="fig">Fig. 8</xref>B–C), whereas in the previously proposed model it was closer to a lumenal orientation [<xref rid="bb0080" ref-type="bibr">16</xref>]. The rotational pitch of the residues in the TM domain of this pentameric model were measured individually [<xref rid="bb0175" ref-type="bibr">35</xref>] and compared to those from the computational models A and B [<xref rid="bb0075" ref-type="bibr">15</xref>] (<xref rid="f0040" ref-type="fig">Fig. 8</xref>D). While values for residues 25–27 are closer to model B, the rest of the sequence is similar to model A, except at residue 28 which deviates from both models. For comparison, the rotational pitch close to model A for residues in E<sub>TM</sub> obtained previously by NMR in DPC micelles [<xref rid="bb0100" ref-type="bibr">20</xref>] is also shown. The present model has been constructed independently from A and B model templates, and the result appears to be a hybrid between the two [<xref rid="bb0075" ref-type="bibr">15</xref>]. This is not surprising since the in silico study assumed a certain rigidity in the TM α-helices [<xref rid="bb0075" ref-type="bibr">15</xref>]. Most of the residues in the model we report have an orientation consistent with model A. This is not surprising, since model A had the lowest energy value for each individual E protein homologs [<xref rid="bb0075" ref-type="bibr">15</xref>]. However, the model gets closer to model B in the turn that contains V25 (<xref rid="f0040" ref-type="fig">Fig. 8</xref>D). This enables V25 to adopt a more interhelical orientation consistent with the revertant mutants that appeared in vivo [<xref rid="bb0150" ref-type="bibr">30</xref>]. Additionally, the helix kink region suggested by infrared dichroism data in lipid bilayers [<xref rid="bb0080" ref-type="bibr">16</xref>] is also observed, which supports the validity of the membrane-mimic environment used herein.<fig id="f0040"><label>Fig. 8</label><caption><p>Orientation of the E<sub>TR</sub> pentamer. (A) Top view and (B) side view of average structure of the E<sub>TR</sub> pentamer bundle in cartoon representation. The N- and C-terminus of one monomeric unit is indicated; (C) top view of a monomer-monomer TM interaction, showing the distances between the side chain of V25 and those of residues appearing in SARS-CoV E V25F revertant mutants [<xref rid="bb0150" ref-type="bibr">30</xref>]; (D) differences in TM residue rotational orientation, ω, between the experimental model proposed here (LMPG) versus computational models A and B [<xref rid="bb0075" ref-type="bibr">15</xref>] and that of E<sub>TM</sub> obtained by NMR in DPC micelles [<xref rid="bb0100" ref-type="bibr">20</xref>]. The region with larger differences between the present model (LMPG) and model A (residues 25–28) is highlighted. (Layout note: 1.5 columns).</p></caption><alt-text id="al0040">Fig. 8</alt-text><graphic xlink:href="gr8_lrg"></graphic></fig></p><p id="p0160">Finally, in LMPG micelles, the C-terminal tail of SARS-CoV E protein is α-helical, more so than observed in mixed DPC/SDS micelles [<xref rid="bb0095" ref-type="bibr">19</xref>], and the presence of extramembrane NOEs suggest interactions between the C-terminal domains that may affect the pentameric conformation.</p></sec></sec><sec id="s0070"><title>Accession numbers</title><p id="p0165">The atomic coordinates have been deposited in the Protein Data Bank (PDB ID: <ext-link ext-link-type="uri" xlink:href="pdb:5X29" id="ir0015">5X29</ext-link>). 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The authors declare no conflict of interest.</p></ack><fn-group><fn id="s0080" fn-type="supplementary-material"><p id="p0175">The <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1016/j.bbamem.2018.02.017" id="ir0020">Transparency document</ext-link> associated with this article can be found, in online version.</p></fn><fn id="s0095" fn-type="supplementary-material"><label>Appendix A</label><p id="p0195">Supplementary data to this article can be found online at <ext-link ext-link-type="uri" xlink:href="https://doi.org/10.1016/j.bbamem.2018.02.017" id="ir0025">https://doi.org/10.1016/j.bbamem.2018.02.017</ext-link>.</p></fn></fn-group></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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