Mechanism of Neomycin and Rev Peptide Binding to the Rev Responsive Element of HIV-1 As Determined by Fluorescence and NMR Spectroscopy
Rev is an essential HIV-1 regulatory protein that binds the Rev responsive element (RRE) within the env gene of the HIV-1 RNA genome and is involved in transport of unspliced or partially spliced viral mRNA from the cell nucleus to the cytoplasm. Previous studies have shown that a short α-helical pe...
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Veröffentlicht in: | Biochemistry (Easton) 2000-05, Vol.39 (19), p.5630-5641 |
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description | Rev is an essential HIV-1 regulatory protein that binds the Rev responsive element (RRE) within the env gene of the HIV-1 RNA genome and is involved in transport of unspliced or partially spliced viral mRNA from the cell nucleus to the cytoplasm. Previous studies have shown that a short α-helical peptide derived from Rev (Rev 34−50), and a truncated form of the RRE sequence provide a useful in vitro system to study this interaction while still preserving the essential aspects of the native complex. We have selectively incorporated the fluorescent probe 2-aminopurine 2‘-O-methylriboside (2-AP) into the RRE sequence in nonperturbing positions (A68 and U72) such that the binding of both Rev peptide and aminoglycoside ligands could be characterized directly by fluorescence methods. Rev peptide binding to the RRE-72AP variant resulted in a 2-fold fluorescence increase that provided a useful signal to monitor this binding interaction (K D = 20 ± 7 nM). Using stopped-flow kinetic measurements, we have shown that specific Rev peptide binding occurs by a two-step process involving diffusion-controlled encounter, followed by isomerization of the RNA. Using the RRE-68AP and -72AP constructs, three classes of binding sites for the aminoglycoside neomycin were unambiguously detected. The first site is noninhibitory to Rev binding (K D = 0.24 ± 0.040 μM), the second site inhibited Rev binding in a competitive fashion (K D = 1.8 ± 0.8 μM), and the third much weaker site (or sites) is attributed to nonspecific binding (K D ≥ 40 μM). Complementary NMR measurements have shown that neomycin forms both a specific binary complex with RRE and a specific ternary complex with RRE and Rev. NMR data further suggest that neomycin occupies a similar high-affinity binding site in both the binary and ternary complexes, and that this site is located in the lower stem region of RRE. |
doi_str_mv | 10.1021/bi992932p |
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Previous studies have shown that a short α-helical peptide derived from Rev (Rev 34−50), and a truncated form of the RRE sequence provide a useful in vitro system to study this interaction while still preserving the essential aspects of the native complex. We have selectively incorporated the fluorescent probe 2-aminopurine 2‘-O-methylriboside (2-AP) into the RRE sequence in nonperturbing positions (A68 and U72) such that the binding of both Rev peptide and aminoglycoside ligands could be characterized directly by fluorescence methods. Rev peptide binding to the RRE-72AP variant resulted in a 2-fold fluorescence increase that provided a useful signal to monitor this binding interaction (K D = 20 ± 7 nM). Using stopped-flow kinetic measurements, we have shown that specific Rev peptide binding occurs by a two-step process involving diffusion-controlled encounter, followed by isomerization of the RNA. Using the RRE-68AP and -72AP constructs, three classes of binding sites for the aminoglycoside neomycin were unambiguously detected. The first site is noninhibitory to Rev binding (K D = 0.24 ± 0.040 μM), the second site inhibited Rev binding in a competitive fashion (K D = 1.8 ± 0.8 μM), and the third much weaker site (or sites) is attributed to nonspecific binding (K D ≥ 40 μM). Complementary NMR measurements have shown that neomycin forms both a specific binary complex with RRE and a specific ternary complex with RRE and Rev. NMR data further suggest that neomycin occupies a similar high-affinity binding site in both the binary and ternary complexes, and that this site is located in the lower stem region of RRE.</description><identifier>ISSN: 0006-2960</identifier><identifier>EISSN: 1520-4995</identifier><identifier>DOI: 10.1021/bi992932p</identifier><identifier>PMID: 10801313</identifier><language>eng</language><publisher>United States: American Chemical Society</publisher><subject>2-Aminopurine - chemistry ; AIDS/HIV ; Amino Acid Sequence ; Anti-Bacterial Agents - metabolism ; Binding, Competitive - genetics ; env gene ; Framycetin - metabolism ; Gene Products, rev - antagonists & inhibitors ; Gene Products, rev - genetics ; Gene Products, rev - metabolism ; HIV-1 - genetics ; HIV-1 - metabolism ; Human immunodeficiency virus 1 ; Kinetics ; Molecular Sequence Data ; neomycin ; Nuclear Magnetic Resonance, Biomolecular ; Peptide Fragments - antagonists & inhibitors ; Peptide Fragments - genetics ; Peptide Fragments - metabolism ; Protein Binding - genetics ; Response Elements ; rev Gene Products, Human Immunodeficiency Virus ; Rev protein ; rev-responsive element ; Spectrometry, Fluorescence ; Thermodynamics</subject><ispartof>Biochemistry (Easton), 2000-05, Vol.39 (19), p.5630-5641</ispartof><rights>Copyright © 2000 American Chemical Society</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-a446t-dde8748bd9cadeec6c5a09ba0bc02ecfd1afb76476d562d31a53eded3a6ec2623</citedby><cites>FETCH-LOGICAL-a446t-dde8748bd9cadeec6c5a09ba0bc02ecfd1afb76476d562d31a53eded3a6ec2623</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://pubs.acs.org/doi/pdf/10.1021/bi992932p$$EPDF$$P50$$Gacs$$H</linktopdf><linktohtml>$$Uhttps://pubs.acs.org/doi/10.1021/bi992932p$$EHTML$$P50$$Gacs$$H</linktohtml><link.rule.ids>314,780,784,2765,27076,27924,27925,56738,56788</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/10801313$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Lacourciere, Karen A</creatorcontrib><creatorcontrib>Stivers, James T</creatorcontrib><creatorcontrib>Marino, John P</creatorcontrib><title>Mechanism of Neomycin and Rev Peptide Binding to the Rev Responsive Element of HIV-1 As Determined by Fluorescence and NMR Spectroscopy</title><title>Biochemistry (Easton)</title><addtitle>Biochemistry</addtitle><description>Rev is an essential HIV-1 regulatory protein that binds the Rev responsive element (RRE) within the env gene of the HIV-1 RNA genome and is involved in transport of unspliced or partially spliced viral mRNA from the cell nucleus to the cytoplasm. Previous studies have shown that a short α-helical peptide derived from Rev (Rev 34−50), and a truncated form of the RRE sequence provide a useful in vitro system to study this interaction while still preserving the essential aspects of the native complex. We have selectively incorporated the fluorescent probe 2-aminopurine 2‘-O-methylriboside (2-AP) into the RRE sequence in nonperturbing positions (A68 and U72) such that the binding of both Rev peptide and aminoglycoside ligands could be characterized directly by fluorescence methods. Rev peptide binding to the RRE-72AP variant resulted in a 2-fold fluorescence increase that provided a useful signal to monitor this binding interaction (K D = 20 ± 7 nM). Using stopped-flow kinetic measurements, we have shown that specific Rev peptide binding occurs by a two-step process involving diffusion-controlled encounter, followed by isomerization of the RNA. Using the RRE-68AP and -72AP constructs, three classes of binding sites for the aminoglycoside neomycin were unambiguously detected. The first site is noninhibitory to Rev binding (K D = 0.24 ± 0.040 μM), the second site inhibited Rev binding in a competitive fashion (K D = 1.8 ± 0.8 μM), and the third much weaker site (or sites) is attributed to nonspecific binding (K D ≥ 40 μM). Complementary NMR measurements have shown that neomycin forms both a specific binary complex with RRE and a specific ternary complex with RRE and Rev. NMR data further suggest that neomycin occupies a similar high-affinity binding site in both the binary and ternary complexes, and that this site is located in the lower stem region of RRE.</description><subject>2-Aminopurine - chemistry</subject><subject>AIDS/HIV</subject><subject>Amino Acid Sequence</subject><subject>Anti-Bacterial Agents - metabolism</subject><subject>Binding, Competitive - genetics</subject><subject>env gene</subject><subject>Framycetin - metabolism</subject><subject>Gene Products, rev - antagonists & inhibitors</subject><subject>Gene Products, rev - genetics</subject><subject>Gene Products, rev - metabolism</subject><subject>HIV-1 - genetics</subject><subject>HIV-1 - metabolism</subject><subject>Human immunodeficiency virus 1</subject><subject>Kinetics</subject><subject>Molecular Sequence Data</subject><subject>neomycin</subject><subject>Nuclear Magnetic Resonance, Biomolecular</subject><subject>Peptide Fragments - antagonists & inhibitors</subject><subject>Peptide Fragments - genetics</subject><subject>Peptide Fragments - metabolism</subject><subject>Protein Binding - genetics</subject><subject>Response Elements</subject><subject>rev Gene Products, Human Immunodeficiency Virus</subject><subject>Rev protein</subject><subject>rev-responsive element</subject><subject>Spectrometry, Fluorescence</subject><subject>Thermodynamics</subject><issn>0006-2960</issn><issn>1520-4995</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2000</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqF0ctu1DAUBmALgei0sOAFkDcgdRHwJXaSZSktU2lmqKYDCzaWY59Ql8QOsVMxT8BrkzZVxQKJlWWdT78vP0KvKHlHCaPva1dVrOKsf4IWVDCS5VUlnqIFIURmrJLkAB3GeDNtc1Lkz9EBJSWhnPIF-r0Gc629ix0ODd5A6PbGeay9xVu4xZfQJ2cBf3DeOv8dp4DTNdyPthD74KO7BXzWQgc-3SUsL75mFJ9E_BESDJ3zYHG9x-ftGAaIBryB-_DNeouvejBpCNGEfv8CPWt0G-Hlw3qEvpyf7U6X2erzp4vTk1Wm81ymzFooi7ysbWW0BTDSCE2qWpPaEAamsVQ3dSHzQlohmeVUCw4WLNcSDJOMH6G3c24_hJ8jxKQ6N12rbbWHMEZVUEo4F_y_kBZCFCUtJ3g8QzM9JQ7QqH5wnR72ihJ1V496rGeyrx9Cx7oD-5ec-5hANgMXE_x6nOvhh5IFL4TaXV6p3Xq1XS2_bdR68m9mr01UN2Ec_PR5_zj4D7jTp2A</recordid><startdate>20000516</startdate><enddate>20000516</enddate><creator>Lacourciere, Karen A</creator><creator>Stivers, James T</creator><creator>Marino, John P</creator><general>American Chemical Society</general><scope>BSCLL</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7TM</scope><scope>7U9</scope><scope>H94</scope><scope>7X8</scope></search><sort><creationdate>20000516</creationdate><title>Mechanism of Neomycin and Rev Peptide Binding to the Rev Responsive Element of HIV-1 As Determined by Fluorescence and NMR Spectroscopy</title><author>Lacourciere, Karen A ; Stivers, James T ; Marino, John P</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a446t-dde8748bd9cadeec6c5a09ba0bc02ecfd1afb76476d562d31a53eded3a6ec2623</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2000</creationdate><topic>2-Aminopurine - chemistry</topic><topic>AIDS/HIV</topic><topic>Amino Acid Sequence</topic><topic>Anti-Bacterial Agents - metabolism</topic><topic>Binding, Competitive - genetics</topic><topic>env gene</topic><topic>Framycetin - metabolism</topic><topic>Gene Products, rev - antagonists & inhibitors</topic><topic>Gene Products, rev - genetics</topic><topic>Gene Products, rev - metabolism</topic><topic>HIV-1 - genetics</topic><topic>HIV-1 - metabolism</topic><topic>Human immunodeficiency virus 1</topic><topic>Kinetics</topic><topic>Molecular Sequence Data</topic><topic>neomycin</topic><topic>Nuclear Magnetic Resonance, Biomolecular</topic><topic>Peptide Fragments - antagonists & inhibitors</topic><topic>Peptide Fragments - genetics</topic><topic>Peptide Fragments - metabolism</topic><topic>Protein Binding - genetics</topic><topic>Response Elements</topic><topic>rev Gene Products, Human Immunodeficiency Virus</topic><topic>Rev protein</topic><topic>rev-responsive element</topic><topic>Spectrometry, Fluorescence</topic><topic>Thermodynamics</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Lacourciere, Karen A</creatorcontrib><creatorcontrib>Stivers, James T</creatorcontrib><creatorcontrib>Marino, John P</creatorcontrib><collection>Istex</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Biochemistry (Easton)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Lacourciere, Karen A</au><au>Stivers, James T</au><au>Marino, John P</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Mechanism of Neomycin and Rev Peptide Binding to the Rev Responsive Element of HIV-1 As Determined by Fluorescence and NMR Spectroscopy</atitle><jtitle>Biochemistry (Easton)</jtitle><addtitle>Biochemistry</addtitle><date>2000-05-16</date><risdate>2000</risdate><volume>39</volume><issue>19</issue><spage>5630</spage><epage>5641</epage><pages>5630-5641</pages><issn>0006-2960</issn><eissn>1520-4995</eissn><abstract>Rev is an essential HIV-1 regulatory protein that binds the Rev responsive element (RRE) within the env gene of the HIV-1 RNA genome and is involved in transport of unspliced or partially spliced viral mRNA from the cell nucleus to the cytoplasm. Previous studies have shown that a short α-helical peptide derived from Rev (Rev 34−50), and a truncated form of the RRE sequence provide a useful in vitro system to study this interaction while still preserving the essential aspects of the native complex. We have selectively incorporated the fluorescent probe 2-aminopurine 2‘-O-methylriboside (2-AP) into the RRE sequence in nonperturbing positions (A68 and U72) such that the binding of both Rev peptide and aminoglycoside ligands could be characterized directly by fluorescence methods. Rev peptide binding to the RRE-72AP variant resulted in a 2-fold fluorescence increase that provided a useful signal to monitor this binding interaction (K D = 20 ± 7 nM). Using stopped-flow kinetic measurements, we have shown that specific Rev peptide binding occurs by a two-step process involving diffusion-controlled encounter, followed by isomerization of the RNA. Using the RRE-68AP and -72AP constructs, three classes of binding sites for the aminoglycoside neomycin were unambiguously detected. The first site is noninhibitory to Rev binding (K D = 0.24 ± 0.040 μM), the second site inhibited Rev binding in a competitive fashion (K D = 1.8 ± 0.8 μM), and the third much weaker site (or sites) is attributed to nonspecific binding (K D ≥ 40 μM). Complementary NMR measurements have shown that neomycin forms both a specific binary complex with RRE and a specific ternary complex with RRE and Rev. NMR data further suggest that neomycin occupies a similar high-affinity binding site in both the binary and ternary complexes, and that this site is located in the lower stem region of RRE.</abstract><cop>United States</cop><pub>American Chemical Society</pub><pmid>10801313</pmid><doi>10.1021/bi992932p</doi><tpages>12</tpages></addata></record> |
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subjects | 2-Aminopurine - chemistry AIDS/HIV Amino Acid Sequence Anti-Bacterial Agents - metabolism Binding, Competitive - genetics env gene Framycetin - metabolism Gene Products, rev - antagonists & inhibitors Gene Products, rev - genetics Gene Products, rev - metabolism HIV-1 - genetics HIV-1 - metabolism Human immunodeficiency virus 1 Kinetics Molecular Sequence Data neomycin Nuclear Magnetic Resonance, Biomolecular Peptide Fragments - antagonists & inhibitors Peptide Fragments - genetics Peptide Fragments - metabolism Protein Binding - genetics Response Elements rev Gene Products, Human Immunodeficiency Virus Rev protein rev-responsive element Spectrometry, Fluorescence Thermodynamics |
title | Mechanism of Neomycin and Rev Peptide Binding to the Rev Responsive Element of HIV-1 As Determined by Fluorescence and NMR Spectroscopy |
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