I(kappa)B(gamma) inhibits DNA binding of NF-kappaB p50 homodimers by interacting with residues that contact DNA.
NF-(kappa)B is an inducible transcription factor that activates many cellular genes involved in stress and immune response and whose DNA binding activity and cellular distribution are regulated by I(kappa)B inhibitor proteins. The interaction between NF-(kappa)B p50 and DNA was investigated by protein footprinting using chemical modification and partial proteolysis. Both methods confirmed lysine-DNA contacts already found in the crystal structure (K-147, K-149, K-244, K-275, and K-278) but also revealed an additional contact in the lysine cluster K-77-K-78-K-80 which was made on an extended DNA. Molecular modelling of such a DNA-protein complex revealed that lysine 80 is ideally placed to make phosphate backbone contacts in the extended DNA. Thus, it seems likely that the entire AB loop, containing lysines 77, 78, and 80, forms a C-shaped clamp that closes around the DNA recognition site. The same protein footprinting approaches were used to probe the interaction of p50 with the ankyrin repeat containing proteins I(kappa)B(gamma) and I(kappa)B(alpha). Lysine residues in p50 that were protected from modification by DNA were also protected from modification by I(kappa)B(gamma) but not I(kappa)B(alpha). Similarly, proteolytic cleavage at p50 residues which contact DNA was inhibited by bound I(kappa)B(gamma) but was enhanced by the presence of I(kappa)B(alpha). Thus, I(kappa)B(gamma) inhibits the DNA binding activity of p50 by direct interactions with residues contacting DNA, whereas the same residues remain exposed in the presence of I(kappa)B(alpha), which binds to p50 but does not block DNA binding.
School of Biological and Medical Sciences, University of St. Andrews, Fife, Scotland.
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S
Bell
J R
JR
Matthews
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Jaffray
R T
RT
Hay
D000595:Amino Acid Sequence; D001482:Base Composition; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D000939:Epitopes; D008239:Lysine; D008958:Models, Molecular; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000737:chemistry; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism
D000939:Epitopes; D016328:NF-kappa B; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014157:Transcription Factors; D004247:DNA; D008239:Lysine