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J. Biol. Chem., Vol. 283, Issue 9, 5632-5641, February 29, 2008

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Subunit-specific Protein Footprinting Reveals Significant Structural Rearrangements and a Role for N-terminal Lys-14 of HIV-1 Integrase during Viral DNA Binding^*

Zhuojun Zhao¹, Christopher J. McKee¹, Jacques J. Kessl, Webster L. Santos², Janet E. Daigle^¶, Alan Engelman^¶, Gregory Verdine, and Mamuka Kvaratskhelia³

From the Center for Retrovirus Research and Comprehensive Cancer Center, College of Pharmacy, The Ohio State University, Columbus, Ohio 43210, the Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, and the ^¶Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute and Division of AIDS, Harvard Medical School, Boston, Massachusetts 02115

To identify functional contacts between HIV-1 integrase (IN) and its viral DNA substrate, we devised a new experimental strategy combining the following two methodologies. First, disulfide-mediated cross-linking was used to site-specifically link select core and C-terminal domain amino acids to respective positions in viral DNA. Next, surface topologies of free IN and IN-DNA complexes were compared using Lys- and Arg-selective small chemical modifiers and mass spectrometric analysis. This approach enabled us to dissect specific contacts made by different monomers within the multimeric complex. The foot-printing studies for the first time revealed the importance of a specific N-terminal domain residue, Lys-14, in viral DNA binding. In addition, a DNA-induced conformational change involving the connection between the core and C-terminal domains was observed. Site-directed mutagenesis experiments confirmed the importance of the identified contacts for recombinant IN activities and virus infection. These new findings provided major constraints, enabling us to identify the viral DNA binding channel in the active full-length IN multimer. The experimental approach described here has general application to mapping interactions within functional nucleoprotein complexes.

Received for publication, June 26, 2007 , and in revised form, November 30, 2007.

^* This work was supported in part by NIAID, National Institutes of Health Grants AI062520 (to M. K.) and AI039394 (to A. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ Both authors contributed equally to this work.

² Present address: Dept. of Chemistry, Virginia Tech, Blacksburg, VA 24061.

³ To whom correspondence should be addressed: The Ohio State University, 500 W. 12th Ave., 238 L.M. Parks Hall, Columbus, OH 43210. Tel.: 614-292-6091; Fax: 614-292-7766; E-mail: kvaratskhelia.1{at}osu.edu.

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