IκBα functions through direct contacts with the nuclear localization signals and the DNA binding sequences of NF-κB

We have determined the binding energies of complexes formed between I kappa B alpha and the wild type and mutational variants of three different Rel/NF-kappa B dimers, namely, the p50/p65 heterodimer and homodimers of p50 and p65. We show that although a common mode of interaction exists between the Rel/NF-kappa B dimers and I kappa B alpha, I kappa B alpha binds the NF-kappa B p50/p65 heterodimer with 60- and 27-fold higher affinity than the p50 and p65 homodimers, respectively. Each of the three flexibly linked segments of the rel homology region of Rel/NF-kappa B proteins (the nuclear localization sequence, the dimerization domain, and the amino-terminal DNA binding domain) is directly engaged in forming the protein/protein interface with the ankyrin repeats and the carboxyl-terminal acidic tail/PEST sequence of I kappa B alpha. In the cell, I kappa B alpha functions to retain NF-kappa B in the cytoplasm and inhibit its DNA binding activity. These properties are a result of the direct involvement of the nuclear localization sequences and of the DNA binding region of NF-kappa B in complex with I kappa B alpha. A model of the interactions in the complex is proposed based on our observations and the crystal structures of Rel/NF-kappa B dimers and the ankyrin domains of related proteins.