Determinants of Mg2+-dependent activities of recombinant human immunodeficiency virus type 1 integrase

The relationship between Mg2+-dependent activity and the self-assembly state of HIV-1 integrase was investigated using different protein preparations. The first preparations, INCHAPS and INdial, were purified in the presence of detergent, but in the case of INdial, the detergent was removed during a final dialysis. The third preparation, IN,,, was purified without any detergent. The three preparations displayed comparable Mn2+-dependent activities. In contrast, the Mg2+-dependent activity that reflects a more realistic view of the physiological activity strongly depended on the preparation. INCHAPS was not capable of using Mg2+ as a cofactor, whereas INzn 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 INCHAPS was monomeric at the concentration of enzymatic assays. Here, we show that LN,, was homogeneously tetrameric under similar conditions. Moreover, INdial that exhibited an intermediary Mg2+-dependent activity existed in a monomer-multimer equilibrium. The level. of Mg2+- but not Mn2+-dependent activity of INdial 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 Mg2+ as a cofactor is related to its self-assembly state in solution, whereas Mn2+-dependent activity is not. Finally, the oligomeric INzn was capable of binding efficiently to DNA regardless of the cationic cofactor, whereas the monomeric INCHAPS strictly required Mn2+. Thus, we propose that a specific conformation of integrase is a prerequisite for its binding to DNA in the presence of Mg2+.