Determinants of Mg²⁺-Dependent Activities of Recombinant Human Immunodeficiency Virus Type 1 Integrase

Abstract

The relationship between Mg(2+)-dependent activity and the self-assembly state of HIV-1 integrase was investigated using different protein preparations. The first preparations, IN(CHAPS) and IN(dial), were purified in the presence of detergent, but in the case of IN(dial), the detergent was removed during a final dialysis. The third preparation, IN(zn), was purified without any detergent. The three preparations displayed comparable Mn(2+)-dependent activities. In contrast, the Mg(2+)-dependent activity that reflects a more realistic view of the physiological activity strongly depended on the preparation. IN(CHAPS) was not capable of using Mg(2+) as a cofactor, whereas IN(zn) was highly active under the same conditions. In the accompanying paper [Deprez, E., et al. (2000) Biochemistry 39, 9275-9284], we used time-resolved fluorescence anisotropy to demonstrate that IN(CHAPS) was monomeric at the concentration of enzymatic assays. Here, we show that IN(zn) was homogeneously tetrameric under similar conditions. Moreover, IN(dial) that exhibited an intermediary Mg(2+)-dependent activity existed in a monomer-multimer equilibrium. The level of Mg(2+)- but not Mn(2+)-dependent activity of IN(dial) was altered by addition of detergent which plays a detrimental role in the maintenance of the oligomeric organization. Our results indicate that the ability of integrase to use Mg(2+) as a cofactor is related to its self-assembly state in solution, whereas Mn(2+)-dependent activity is not. Finally, the oligomeric IN(zn) was capable of binding efficiently to DNA regardless of the cationic cofactor, whereas the monomeric IN(CHAPS) strictly required Mn(2+). Thus, we propose that a specific conformation of integrase is a prerequisite for its binding to DNA in the presence of Mg(2+).

DNA binding induces dissociation of the multimeric form of HIV-1 integrase: A time-resolved fluorescence anisotropy study

“…HIV-1 IN was purified as described previously under native conditions (7). Briefly, His-tagged IN containing a thrombin cleavage site adjacent to the His tag was overexpressed in Escherichia coli BL21(DE3).…”

“…When purified in the presence of detergent, IN has been found to be monomeric at submicromolar concentration and active only in the presence of Mn 2ϩ . In contrast, when purified in the absence of detergent and in the presence of zinc, two conditions that favor the multimerization process (7,(15)(16)(17), IN has been found to be mainly tetrameric and highly active in the presence of Mg 2ϩ .In this work, we investigated the oligomeric state of the Mg 2ϩ -competent IN bound to the viral DNA under conditions typically used in in vitro enzymatic assays, i.e., 100-200 nM IN͞20 nM substrate DNA. Time-resolved fluorescence anisotropy (TFA) is suitable for such a study because its sensitivity allows detection of low protein or DNA concentrations, and the rotational correlation time ( ) as determined by TFA is directly related to the apparent volume of macromolecules or complexes.…”

“…When purified in the presence of detergent, IN has been found to be monomeric at submicromolar concentration and active only in the presence of Mn 2ϩ . In contrast, when purified in the absence of detergent and in the presence of zinc, two conditions that favor the multimerization process (7,(15)(16)(17), IN has been found to be mainly tetrameric and highly active in the presence of Mg 2ϩ .…”

“…Specific protein-DNA contacts occur preferentially in the presence of Mg 2ϩ (5), and more nonspecific cleavage is detected in the presence of Mn 2ϩ (6). The transfer pattern on the target DNA also depends on the nature of the cation (7). Consequently, the efficiency of inhibitors can be different by using either Mg 2ϩ or Mn 2ϩ in assays (8).…”

“…Furthermore, it is strongly believed that self-association of IN plays a key role in governing the enzyme function (13)(14)(15)(16). Recently, we have shown that the capability of IN to use Mg 2ϩ is related to the protein self-association in solution (7,17). When purified in the presence of detergent, IN has been found to be monomeric at submicromolar concentration and active only in the presence of Mn 2ϩ .…”