Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 283, Issue 9, Pages 5632-5641Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M705241200
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Funding
- NIAID NIH HHS [AI062520, AI039394, R01 AI062520, R01 AI039394, R37 AI039394] Funding Source: Medline
- NIGMS NIH HHS [R01 GM044853-15, F32 GM067380, R01 GM044853, F32 GM067380-03] Funding Source: Medline
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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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