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

Abstract

We have determined the binding energies of complexes formed between IκBα and the wild type and mutational variants of three different Rel/NF- κB 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- κB dimers and IκBα, IκBα binds the NF-κB 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-κB 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 IκBα. In the cell, IκBα functions to retain NF-κB 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-κB in complex with IκBα. A model of the interactions in the complex is proposed based on our observations and the crystal structures of Rel/NF-κB dimers and the ankyrin domains of related proteins.