I-KAPPA-B-GAMMA INHIBITS DNA-BINDING OF NF-KAPPA-B P50 HOMODIMERS BY INTERACTING WITH RESIDUES THAT CONTACT DNA

NF-kappa B is an inducible transcription factor that activates many ce llular genes involved in stress and immune response and whose DNA bind ing activity and cellular distribution are regulated by I kappa B inhi bitor proteins. The interaction between NF-kappa B p50 and DNA was inv estigated by protein footprinting using chemical modification and part ial proteolysis. Both methods confirmed lysine-DNA contacts already fo und in the crystal structure (K-147, K-149, K-244, K-275, and K-278) b ut 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 likel y 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 p rotein footprinting approaches were used to probe the interaction of p ig with the ankyrin repeat containing proteins I kappa B gamma and I k appa B alpha. Lysine residues in p50 that were protected from modifica tion by DNA were also protected from modification by I kappa B gamma b ut not I kappa B alpha. Similarly, proteolytic cleavage at p50 residue s which contact DNA was inhibited by bound I kappa B gamma but was enh anced by the presence of I kappa B alpha. Thus, I kappa B gamma inhibi ts the DNA binding activity of p50 by direct interactions with residue s contacting DNA, whereas the same residues remain exposed in the pres ence of I kappa B alpha, which binds to p50 but does not block DNA bin ding.