Dynamic, Thermodynamic, and Kinetic Basis for Recognition and Transformation of DNA by Human Immunodeficiency Virus Type 1 Integrase†
Identifieur interne : 003245 ( Main/Exploration ); précédent : 003244; suivant : 003246Dynamic, Thermodynamic, and Kinetic Basis for Recognition and Transformation of DNA by Human Immunodeficiency Virus Type 1 Integrase†
Auteurs : Dmitrii V. Bugreev [Italie] ; Svetlana Baranova [Italie] ; Olga D. Zakharova [Italie] ; Vincent Parissi [Italie] ; Cécile Desjobert [Italie] ; Enzo Sottofattori [Italie] ; Alessandro Balbi [Italie] ; Simon Litvak [Italie] ; Laura Tarrago-Litvak [Italie] ; Georgy A. Nevinsky [Italie]Source :
- Biochemistry [ 0006-2960 ] ; 2003.
Abstract
Specific interactions between retroviral integrase (IN) and long terminal repeats are required for insertion of viral DNA into the host genome. To characterize quantitatively the determinants of substrate specificity, we used a method based on a stepwise increase in ligand complexity. This allowed an estimation of the relative contributions of each nucleotide from oligonucleotides to the total affinity for IN. The interaction of HIV-1 integrase with specific (containing sequences from the LTR) or nonspecific oligonucleotides was analyzed using a thermodynamic model. Integrase interacted with oligonucleotides through a superposition of weak contacts with their bases, and more importantly, with the internucleotide phosphate groups. All these structural components contributed in a combined way to the free energy of binding with the major contribution made by the conserved 3‘-terminal GT, and after its removal, by the CA dinucleotide. In contrast to nonspecific oligonucleotides that inhibited the reaction catalyzed by IN, specific oligonucleotides enhanced the activity, probably owing to the effect of sequence-specific ligands on the dynamic equilibrium between the oligomeric forms of IN. However, after preactivation of IN by incubation with Mn2+, the specific oligonucleotides were also able to inhibit the processing reaction. We found that nonspecific interactions of IN with DNA provide ∼8 orders of magnitude in the affinity (ΔG° ≈ −10.3 kcal/mol), while the relative contribution of specific nucleotides of the substrate corresponds to ∼1.5 orders of magnitude (ΔG° ≈ − 2.0 kcal/mol). Formation of the Michaelis complex between IN and specific DNA cannot by itself account for the major contribution of enzyme specificity, which lies in the kcat term; the rate is increased by more than 5 orders of magnitude upon transition from nonspecific to specific oligonucleotides.
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DOI: 10.1021/bi0300480
Affiliations:
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Nevinsky</name><affiliation wicri:level="1"><country xml:lang="fr">Italie</country><wicri:regionArea>Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 8 Lavrentieva Avenue,Novosibirsk 630090, Russia, UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat,33076 Bordeaux Cedex, France and IFR 66 Bordeaux, France, and University of Genoa, Department of PharmaceuticalSciences, Viale Benedetto XV, 3, Genoa-3</wicri:regionArea><wicri:noRegion>Genoa-3</wicri:noRegion></affiliation><affiliation></affiliation><affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j" type="main">Biochemistry</title><title level="j" type="abbrev">Biochemistry</title><idno type="ISSN">0006-2960</idno><idno type="eISSN">1520-4995</idno><imprint><publisher>American Chemical Society</publisher><date type="e-published">2003</date><date type="published">2003</date><biblScope unit="vol">42</biblScope><biblScope unit="issue">30</biblScope><biblScope unit="page" from="9235">9235</biblScope><biblScope unit="page" to="9247">9247</biblScope></imprint><idno type="ISSN">0006-2960</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0006-2960</idno></seriesStmt></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract">Specific interactions between retroviral integrase (IN) and long terminal repeats are required for insertion of viral DNA into the host genome. To characterize quantitatively the determinants of substrate specificity, we used a method based on a stepwise increase in ligand complexity. This allowed an estimation of the relative contributions of each nucleotide from oligonucleotides to the total affinity for IN. The interaction of HIV-1 integrase with specific (containing sequences from the LTR) or nonspecific oligonucleotides was analyzed using a thermodynamic model. Integrase interacted with oligonucleotides through a superposition of weak contacts with their bases, and more importantly, with the internucleotide phosphate groups. All these structural components contributed in a combined way to the free energy of binding with the major contribution made by the conserved 3‘-terminal GT, and after its removal, by the CA dinucleotide. In contrast to nonspecific oligonucleotides that inhibited the reaction catalyzed by IN, specific oligonucleotides enhanced the activity, probably owing to the effect of sequence-specific ligands on the dynamic equilibrium between the oligomeric forms of IN. However, after preactivation of IN by incubation with Mn2+, the specific oligonucleotides were also able to inhibit the processing reaction. We found that nonspecific interactions of IN with DNA provide ∼8 orders of magnitude in the affinity (ΔG° ≈ −10.3 kcal/mol), while the relative contribution of specific nucleotides of the substrate corresponds to ∼1.5 orders of magnitude (ΔG° ≈ − 2.0 kcal/mol). Formation of the Michaelis complex between IN and specific DNA cannot by itself account for the major contribution of enzyme specificity, which lies in the kcat term; the rate is increased by more than 5 orders of magnitude upon transition from nonspecific to specific oligonucleotides.</div></front></TEI><affiliations><list><country><li>Italie</li></country></list><tree><country name="Italie"><noRegion><name sortKey="Bugreev, Dmitrii V" sort="Bugreev, Dmitrii V" uniqKey="Bugreev D" first="Dmitrii V." last="Bugreev">Dmitrii V. Bugreev</name></noRegion><name sortKey="Balbi, Alessandro" sort="Balbi, Alessandro" uniqKey="Balbi A" first="Alessandro" last="Balbi">Alessandro Balbi</name><name sortKey="Baranova, Svetlana" sort="Baranova, Svetlana" uniqKey="Baranova S" first="Svetlana" last="Baranova">Svetlana Baranova</name><name sortKey="Desjobert, Cecile" sort="Desjobert, Cecile" uniqKey="Desjobert C" first="Cécile" last="Desjobert">Cécile Desjobert</name><name sortKey="Litvak, Simon" sort="Litvak, Simon" uniqKey="Litvak S" first="Simon" last="Litvak">Simon Litvak</name><name sortKey="Nevinsky, Georgy A" sort="Nevinsky, Georgy A" uniqKey="Nevinsky G" first="Georgy A." last="Nevinsky">Georgy A. Nevinsky</name><name sortKey="Parissi, Vincent" sort="Parissi, Vincent" uniqKey="Parissi V" first="Vincent" last="Parissi">Vincent Parissi</name><name sortKey="Sottofattori, Enzo" sort="Sottofattori, Enzo" uniqKey="Sottofattori E" first="Enzo" last="Sottofattori">Enzo Sottofattori</name><name sortKey="Tarrago Litvak, Laura" sort="Tarrago Litvak, Laura" uniqKey="Tarrago Litvak L" first="Laura" last="Tarrago-Litvak">Laura Tarrago-Litvak</name><name sortKey="Zakharova, Olga D" sort="Zakharova, Olga D" uniqKey="Zakharova O" first="Olga D." last="Zakharova">Olga D. Zakharova</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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