Dynamic, Thermodynamic, and Kinetic Basis for Recognition and Transformation of DNA by Human Immunodeficiency Virus Type 1 Integrase

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 Mn(2+), the specific oligonucleotides were also able to inhibit the processing reaction. We found that nonspecific interactions of IN with DNA provide approximately 8 orders of magnitude in the affinity (Delta G degrees approximately equal to -10.3 kcal/mol), while the relative contribution of specific nucleotides of the substrate corresponds to approximately 1.5 orders of magnitude (Delta G degrees approximately equal to - 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 k(cat) term; the rate is increased by more than 5 orders of magnitude upon transition from nonspecific to specific oligonucleotides.

Biochemical Analysis of HIV-1 Integrase Variants Resistant to Strand Transfer Inhibitors

“…This staging allows an accurate measurement of the kinetics also providing the K m for target utilization. The K m value (14.2 Ϯ 1.3 nM) for the WT enzyme for target DNA utilization in the strand transfer reaction is similar to the reported dissociation constant (K d ) for double-stranded DNA binding to integrase using BIAcore measurements (23 Ϯ 2 nM) (39) and slightly higher than that reported for the association of a fluorescein-labeled oligoduplex by fluorescence anisotropy changes (35 nM) (40) or the inhibition constant (K i ) for the binding of a nonspecific oligoduplex DNA to integrase measured by inhibition of 3Ј-processing (40 nM) (41). This is evidence that the approach taken here of using a two-phase system to measure the kinetics is valid.…”

Designed zinc finger protein interacting with the HIV‐1 integrase recognition sequence at 2‐LTR‐circle junctions

“…The binding activity was on the nanomolar scale, which is similar to the affinity level of HIV-1 IN. 32,33 This finding suggested that the HIV-1 IN might be interfered with by the 2LTRZFP as well. The specificity of this binding was Figure 8.…”

Identification by Phage Display Selection of a Short Peptide Able To Inhibit Only the Strand Transfer Reaction Catalyzed by Human Immunodeficiency Virus Type 1 Integrase

“…Recent studies support the possible existence of different IN conformations or distinct sites needed in the recognition of the viral and the cellular target DNAs (5,(35)(36)(37). In parallel, a thermodynamical model based on the SILC (stepwise increase in ligand complexity) method (38) also suggests an organization of the IN active site that explains specific binding of the viral and target substrates through the different steps of catalysis. The characterization of substrates binding to IN showed a relatively higher enzyme affinity for the 3′-terminal CA processed ODN than that for 3′-terminal GT nonprocessed substrate, suggesting that different subsites of IN form specific contacts with the 3′-terminal dinucleotide of viral DNA and target DNA (38).…”