Mapping DNA-binding sites of HIV-1 integrase by protein footprinting.
The HIV-1 integrase protein catalyzes integration of the viral genome into host cell DNA. Whereas the structures of the three domains of integrase have been solved separately, both the structural organization of the full-length protein and its interaction with DNA remain unresolved. A protein footprinting approach was employed to investigate the accessibility of residues in the full-length soluble integrase mutant, INF(185K,C280S), to proteolytic attack in the absence and presence of DNA. The N-terminal and C-terminal domains were relatively more accessible to proteolytic attack than the core domain. The susceptibility to proteolytic attack was specifically affected by DNA at residues Lys34, in the N-terminal domain, Lys111, Lys136, Glu138, Lys156-Lys160, Lys185-Lys188, in the core domain, and Asp207, Lys 215, Glu246, Lys258 and Lys273 in the linker and C-terminal domain, suggesting that these regions are involved in, or shielded by, DNA binding. Lys34 is positioned in a putative dimerization domain, consistent with the notion that DNA stabilizes the dimeric state of integrase.
Dirac
A M
AM
Department of Molecular and Structural Biology, Aarhus University, Denmark.
Kjems
J
J
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D019427:HIV Integrase / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D023302:Protein Footprinting; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
D000596:Amino Acids; D004268:DNA-Binding Proteins; D015345:Oligonucleotide Probes; D011993:Recombinant Fusion Proteins; D004247:DNA; D019427:HIV Integrase