Cross-linking Mass Spectrometry and Mutagenesis Confirm the Functional Importance of Surface Interactions between CYP3A4 and Holo/Apo Cytochrome b5
Identifieur interne : 000423 ( Pmc/Corpus ); précédent : 000422; suivant : 000424Cross-linking Mass Spectrometry and Mutagenesis Confirm the Functional Importance of Surface Interactions between CYP3A4 and Holo/Apo Cytochrome b5
Auteurs : Chunsheng Zhao ; Qiuxia Gao ; Arthur G. Roberts ; Scott A. Shaffer ; Catalin E. Doneanu ; Song Xue ; David R. Goodlett ; Sidney D. Nelson ; William M. AtkinsSource :
- Biochemistry [ 0006-2960 ] ; 2012.
Abstract
Cytochrome
Url:
DOI: 10.1021/bi301069r
PubMed: 23150942
PubMed Central: 3568533
Links to Exploration step
PMC:3568533Le document en format XML
<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>
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<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>
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<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>
<pmc-dir>properties manuscript</pmc-dir>
<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>
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