Subunit specific protein footprinting reveals significant structural rearrangements and a role for N-terminal LYS-14 of HIV-1 integrase during viral DNA binding

doi:10.1074/jbc.M705241200

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Papers In Press, published online ahead of print December 19, 2007
J. Biol. Chem, 10.1074/jbc.M705241200
Submitted on June 26, 2007
Accepted on December 19, 2007

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 Zhao, Christopher J. McKee, Jacques J. Kessl, Webster L. Santos, Janet E. Daigle, Alan Engelman, Gregory Verdine, and Mamuka Kvaratskhelia

College of Pharmacy, The Ohio State University, Columbus, OH 43210

Corresponding Author: kvaratskhelia.1{at}osu.edu

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 footprinting 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.

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