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

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• 作者：Hervé ; Leh ; Priscille Brodin ; Julien Bischerour ; Eric Deprez ; Patrick Tauc ; Jean-Claude Brochon ; Eric LeCam ; Dominique Coulaud ; Christian Auclair ; and Jean-Franç ; ois Mouscadet
• 刊名：Biochemistry
• 出版年：2000
• 出版时间：August 8, 2000
• 年：2000
• 卷：39
• 期：31
• 页码：9285 - 9294
• 全文大小：236K
• 年卷期：v.39,no.31(August 8, 2000)
• ISSN：1520-4995

文摘

The relationship between Mg²⁺-dependent activity and the self-assembly state of HIV-1 integrasewas investigated using different protein preparations. The first preparations, IN_CHAPS and IN_dial, were purifiedin 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 comparableMn²⁺-dependent activities. In contrast, the Mg²⁺-dependent activity that reflects a more realistic view ofthe physiological activity strongly depended on the preparation. IN_CHAPS was not capable of using Mg²⁺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 anisotropyto demonstrate that IN_CHAPS was monomeric at the concentration of enzymatic assays. Here, we showthat IN_zn was homogeneously tetrameric under similar conditions. Moreover, IN_dial that exhibited anintermediary Mg²⁺-dependent activity existed in a monomer-multimer equilibrium. The level of Mg²⁺-but not Mn²⁺-dependent activity of IN_dial was altered by addition of detergent which plays a detrimentalrole in the maintenance of the oligomeric organization. Our results indicate that the ability of integrase touse Mg²⁺ as a cofactor is related to its self-assembly state in solution, whereas Mn²⁺-dependent activityis not. Finally, the oligomeric IN_zn was capable of binding efficiently to DNA regardless of the cationiccofactor, whereas the monomeric IN_CHAPS strictly required Mn²⁺. Thus, we propose that a specificconformation of integrase is a prerequisite for its binding to DNA in the presence of Mg²⁺.