Subunit-specific protein footprinting reveals significant structural rearrangements and a role for N-terminal Lys-14 of HIV-1 Integrase during viral DNA binding.
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.
Zhao
Zhuojun
Z
Center for Retrovirus Research and Comprehensive Cancer Center, College of Pharmacy, The Ohio State University, Columbus, Ohio 43210, USA.
McKee
Christopher J
CJ
Kessl
Jacques J
JJ
Santos
Webster L
WL
Daigle
Janet E
JE
Engelman
Alan
A
Verdine
Gregory
G
Kvaratskhelia
Mamuka
M
D019943:Amino Acid Substitution; D002460:Cell Line; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015658:HIV Infections / Q000201:enzymology* / Q000235:genetics; D019427:HIV Integrase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping / Q000379:methods; D020836:Protein Structure, Quaternary / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology
D004279:DNA, Viral; D019427:HIV Integrase