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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Cross-linking Mass Spectrometry and Mutagenesis Confirm the Functional Importance of Surface Interactions between CYP3A4 and Holo/Apo Cytochrome <italic>b<sub>5</sub></italic></title><author><name sortKey="Zhao, Chunsheng" sort="Zhao, Chunsheng" uniqKey="Zhao C" first="Chunsheng" last="Zhao">Chunsheng Zhao</name><affiliation><nlm:aff id="A1">Department of Medicinal Chemistry, University of Washington, Box 357610, Seattle, Washington 98195</nlm:aff></affiliation></author><author><name sortKey="Gao, Qiuxia" sort="Gao, Qiuxia" uniqKey="Gao Q" first="Qiuxia" last="Gao">Qiuxia Gao</name><affiliation><nlm:aff id="A1">Department of Medicinal Chemistry, University of Washington, Box 357610, Seattle, Washington 98195</nlm:aff></affiliation></author><author><name sortKey="Roberts, Arthur G" sort="Roberts, Arthur G" uniqKey="Roberts A" first="Arthur G." last="Roberts">Arthur G. Roberts</name><affiliation><nlm:aff id="A3">Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, GA 30605</nlm:aff></affiliation></author><author><name sortKey="Shaffer, Scott A" sort="Shaffer, Scott A" uniqKey="Shaffer S" first="Scott A." last="Shaffer">Scott A. Shaffer</name><affiliation><nlm:aff id="A2">Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Shrewsbury, MA 01545</nlm:aff></affiliation></author><author><name sortKey="Doneanu, Catalin E" sort="Doneanu, Catalin E" uniqKey="Doneanu C" first="Catalin E." last="Doneanu">Catalin E. Doneanu</name><affiliation><nlm:aff id="A1">Department of Medicinal Chemistry, University of Washington, Box 357610, Seattle, Washington 98195</nlm:aff></affiliation></author><author><name sortKey="Xue, Song" sort="Xue, Song" uniqKey="Xue S" first="Song" last="Xue">Song Xue</name><affiliation><nlm:aff id="A4">Microsoft Incorporation, 1 Microsoft Way, Redmond, Washington 98052</nlm:aff></affiliation></author><author><name sortKey="Goodlett, David R" sort="Goodlett, David R" uniqKey="Goodlett D" first="David R." last="Goodlett">David R. Goodlett</name><affiliation><nlm:aff id="A1">Department of Medicinal Chemistry, University of Washington, Box 357610, Seattle, Washington 98195</nlm:aff></affiliation></author><author><name sortKey="Nelson, Sidney D" sort="Nelson, Sidney D" uniqKey="Nelson S" first="Sidney D." last="Nelson">Sidney D. Nelson</name><affiliation><nlm:aff id="A1">Department of Medicinal Chemistry, University of Washington, Box 357610, Seattle, Washington 98195</nlm:aff></affiliation></author><author><name sortKey="Atkins, William M" sort="Atkins, William M" uniqKey="Atkins W" first="William M." last="Atkins">William M. Atkins</name><affiliation><nlm:aff id="A1">Department of Medicinal Chemistry, University of Washington, Box 357610, Seattle, Washington 98195</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">23150942</idno><idno type="pmc">3568533</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3568533</idno><idno type="RBID">PMC:3568533</idno><idno type="doi">10.1021/bi301069r</idno><date when="2012">2012</date><idno type="wicri:Area/Pmc/Corpus">000423</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Cross-linking Mass Spectrometry and Mutagenesis Confirm the Functional Importance of Surface Interactions between CYP3A4 and Holo/Apo Cytochrome <italic>b<sub>5</sub></italic></title><author><name sortKey="Zhao, Chunsheng" sort="Zhao, Chunsheng" uniqKey="Zhao C" first="Chunsheng" last="Zhao">Chunsheng Zhao</name><affiliation><nlm:aff id="A1">Department of Medicinal Chemistry, University of Washington, Box 357610, Seattle, Washington 98195</nlm:aff></affiliation></author><author><name sortKey="Gao, Qiuxia" sort="Gao, Qiuxia" uniqKey="Gao Q" first="Qiuxia" last="Gao">Qiuxia Gao</name><affiliation><nlm:aff id="A1">Department of Medicinal Chemistry, University of Washington, Box 357610, Seattle, Washington 98195</nlm:aff></affiliation></author><author><name sortKey="Roberts, Arthur G" sort="Roberts, Arthur G" uniqKey="Roberts A" first="Arthur G." last="Roberts">Arthur G. Roberts</name><affiliation><nlm:aff id="A3">Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, GA 30605</nlm:aff></affiliation></author><author><name sortKey="Shaffer, Scott A" sort="Shaffer, Scott A" uniqKey="Shaffer S" first="Scott A." last="Shaffer">Scott A. Shaffer</name><affiliation><nlm:aff id="A2">Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Shrewsbury, MA 01545</nlm:aff></affiliation></author><author><name sortKey="Doneanu, Catalin E" sort="Doneanu, Catalin E" uniqKey="Doneanu C" first="Catalin E." last="Doneanu">Catalin E. Doneanu</name><affiliation><nlm:aff id="A1">Department of Medicinal Chemistry, University of Washington, Box 357610, Seattle, Washington 98195</nlm:aff></affiliation></author><author><name sortKey="Xue, Song" sort="Xue, Song" uniqKey="Xue S" first="Song" last="Xue">Song Xue</name><affiliation><nlm:aff id="A4">Microsoft Incorporation, 1 Microsoft Way, Redmond, Washington 98052</nlm:aff></affiliation></author><author><name sortKey="Goodlett, David R" sort="Goodlett, David R" uniqKey="Goodlett D" first="David R." last="Goodlett">David R. Goodlett</name><affiliation><nlm:aff id="A1">Department of Medicinal Chemistry, University of Washington, Box 357610, Seattle, Washington 98195</nlm:aff></affiliation></author><author><name sortKey="Nelson, Sidney D" sort="Nelson, Sidney D" uniqKey="Nelson S" first="Sidney D." last="Nelson">Sidney D. Nelson</name><affiliation><nlm:aff id="A1">Department of Medicinal Chemistry, University of Washington, Box 357610, Seattle, Washington 98195</nlm:aff></affiliation></author><author><name sortKey="Atkins, William M" sort="Atkins, William M" uniqKey="Atkins W" first="William M." last="Atkins">William M. Atkins</name><affiliation><nlm:aff id="A1">Department of Medicinal Chemistry, University of Washington, Box 357610, Seattle, Washington 98195</nlm:aff></affiliation></author></analytic><series><title level="j">Biochemistry</title><idno type="ISSN">0006-2960</idno><idno type="eISSN">1520-4995</idno><imprint><date when="2012">2012</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p id="P1">Cytochrome <italic>b<sub>5</sub></italic> (cyt <italic>b<sub>5</sub></italic>) is one of the key components in the microsomal cytochrome P450 monooxygenase system. Consensus has not been reached on the underlying mechanism of cyt <italic>b<sub>5</sub></italic> modulation of CYP catalysis. Both cyt <italic>b<sub>5</sub></italic> and apo <italic>b<sub>5</sub></italic>, are reported to stimulate the activity of several P450 isoforms. In the present study, the surface interactions of both holo and apo <italic>b<sub>5</sub></italic> with CYP3A4 were investigated and compared for the first time. Chemical cross-linking coupled with mass spectrometric analysis was used to identify the potential electrostatic interactions between the protein surfaces. Subsequently, the interaction models of holo/apo <italic>b<sub>5</sub></italic> with CYP3A4 were built using the identified interacting sites as constraints. Both cyt <italic>b<sub>5</sub></italic> and apo <italic>b<sub>5</sub></italic> were predicted to bind to the same groove on CYP3A4 with close contacts to the B-B’ loop of CYP3A4, a substrate recognition site (SRS). Mutagenesis studies further confirmed that the interacting sites on CYP3A4 (Lys96, Lys127 and Lys421) are of functional importance. Mutation of these residues reduced or abolished cyt <italic>b<sub>5</sub></italic> binding affinity. The critical role of Arg446 on CYP3A4 in binding to cyt <italic>b<sub>5</sub></italic> and/or cytochrome P450 reductase (CPR) was also discovered. The results indicated that electrostatic interactions on the interface of the two proteins are functionally important. The results indicate that the apo cyt <italic>b</italic><sub>5</sub> can dock with CYP3A4 in a manner analogous to holo cyt <italic>b</italic><sub>5</sub> so electron transfer from cyt <italic>b</italic><sub>5</sub> is not required for its effects.</p></div></front></TEI><pmc article-type="research-article"><pmc-comment>The publisher of this article does not allow downloading of the full text in XML form.</pmc-comment>
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<front><journal-meta><journal-id journal-id-type="nlm-journal-id">0370623</journal-id><journal-id journal-id-type="pubmed-jr-id">1028</journal-id><journal-id journal-id-type="nlm-ta">Biochemistry</journal-id><journal-id journal-id-type="iso-abbrev">Biochemistry</journal-id><journal-title-group><journal-title>Biochemistry</journal-title></journal-title-group><issn pub-type="ppub">0006-2960</issn><issn pub-type="epub">1520-4995</issn></journal-meta><article-meta><article-id pub-id-type="pmid">23150942</article-id><article-id pub-id-type="pmc">3568533</article-id><article-id pub-id-type="doi">10.1021/bi301069r</article-id><article-id pub-id-type="manuscript">NIHMS421655</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Cross-linking Mass Spectrometry and Mutagenesis Confirm the Functional Importance of Surface Interactions between CYP3A4 and Holo/Apo Cytochrome <italic>b<sub>5</sub></italic></article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Zhao</surname><given-names>Chunsheng</given-names></name><xref ref-type="aff" rid="A1">†</xref></contrib><contrib contrib-type="author"><name><surname>Gao</surname><given-names>Qiuxia</given-names></name><xref ref-type="aff" rid="A1">†</xref></contrib><contrib contrib-type="author"><name><surname>Roberts</surname><given-names>Arthur G.</given-names></name><xref ref-type="aff" rid="A3">‡</xref></contrib><contrib contrib-type="author"><name><surname>Shaffer</surname><given-names>Scott A.</given-names></name><xref ref-type="aff" rid="A2">||</xref></contrib><contrib contrib-type="author"><name><surname>Doneanu</surname><given-names>Catalin E.</given-names></name><xref ref-type="aff" rid="A1">†</xref></contrib><contrib contrib-type="author"><name><surname>Xue</surname><given-names>Song</given-names></name><xref ref-type="aff" rid="A4">§</xref></contrib><contrib contrib-type="author"><name><surname>Goodlett</surname><given-names>David R.</given-names></name><xref ref-type="aff" rid="A1">†</xref></contrib><contrib contrib-type="author"><name><surname>Nelson</surname><given-names>Sidney D.</given-names></name><xref ref-type="aff" rid="A1">†</xref></contrib><contrib contrib-type="author"><name><surname>Atkins</surname><given-names>William M.</given-names></name><xref ref-type="aff" rid="A1">†</xref><xref rid="FN1" ref-type="author-notes">*</xref></contrib></contrib-group><aff id="A1"><label>†</label>Department of Medicinal Chemistry, University of Washington, Box 357610, Seattle, Washington 98195</aff><aff id="A2"><label>||</label>Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Shrewsbury, MA 01545</aff><aff id="A3"><label>‡</label>Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, GA 30605</aff><aff id="A4"><label>§</label>Microsoft Incorporation, 1 Microsoft Way, Redmond, Washington 98052</aff><author-notes><corresp id="FN1"><label>*</label>Corresponding Author: To whom correspondence should be addressed: Telephone: (206) 685-0379. Fax: (206) 685-3252. <email>winky@u.washington.edu</email></corresp></author-notes><pub-date pub-type="nihms-submitted"><day>12</day><month>12</month><year>2012</year></pub-date><pub-date pub-type="epub"><day>14</day><month>11</month><year>2012</year></pub-date><pub-date pub-type="ppub"><day>27</day><month>11</month><year>2012</year></pub-date><pub-date pub-type="pmc-release"><day>27</day><month>11</month><year>2013</year></pub-date><volume>51</volume><issue>47</issue><fpage>9488</fpage><lpage>9500</lpage><abstract><p id="P1">Cytochrome <italic>b<sub>5</sub></italic> (cyt <italic>b<sub>5</sub></italic>) is one of the key components in the microsomal cytochrome P450 monooxygenase system. Consensus has not been reached on the underlying mechanism of cyt <italic>b<sub>5</sub></italic> modulation of CYP catalysis. Both cyt <italic>b<sub>5</sub></italic> and apo <italic>b<sub>5</sub></italic>, are reported to stimulate the activity of several P450 isoforms. In the present study, the surface interactions of both holo and apo <italic>b<sub>5</sub></italic> with CYP3A4 were investigated and compared for the first time. Chemical cross-linking coupled with mass spectrometric analysis was used to identify the potential electrostatic interactions between the protein surfaces. Subsequently, the interaction models of holo/apo <italic>b<sub>5</sub></italic> with CYP3A4 were built using the identified interacting sites as constraints. Both cyt <italic>b<sub>5</sub></italic> and apo <italic>b<sub>5</sub></italic> were predicted to bind to the same groove on CYP3A4 with close contacts to the B-B’ loop of CYP3A4, a substrate recognition site (SRS). Mutagenesis studies further confirmed that the interacting sites on CYP3A4 (Lys96, Lys127 and Lys421) are of functional importance. Mutation of these residues reduced or abolished cyt <italic>b<sub>5</sub></italic> binding affinity. The critical role of Arg446 on CYP3A4 in binding to cyt <italic>b<sub>5</sub></italic> and/or cytochrome P450 reductase (CPR) was also discovered. The results indicated that electrostatic interactions on the interface of the two proteins are functionally important. The results indicate that the apo cyt <italic>b</italic><sub>5</sub> can dock with CYP3A4 in a manner analogous to holo cyt <italic>b</italic><sub>5</sub> so electron transfer from cyt <italic>b</italic><sub>5</sub> is not required for its effects.</p></abstract><kwd-group><kwd>CYP3A4</kwd><kwd>cyt <italic>b<sub>5</sub></italic></kwd><kwd>apo <italic>b<sub>5</sub></italic></kwd><kwd>interaction</kwd><kwd>chemical cross-linking</kwd><kwd>MS</kwd><kwd>mutagenesis</kwd><kwd>PDB: 1TQN</kwd><kwd>1CYO</kwd><kwd>1I87</kwd></kwd-group><funding-group><award-group><funding-source country="United States">National Institute of General Medical Sciences : NIGMS</funding-source><award-id>P01 GM032165 || GM</award-id></award-group></funding-group></article-meta></front></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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