Mechanism of I kappa B alpha binding to NF-kappa B dimers

X-ray crystal structures of the NF-kappa B I kappa B alpha complex revealed an extensive and complex protein-protein interface involving independent s tructural elements present in both I kappa B alpha and NF-kappa B. In this study, we employ a gel electrophoretic mobility shift assay to assess and q uantitate the relative contributions of the observed interactions toward ov erall complex binding affinity. I kappa B alpha preferentially binds to the p50/p65 heterodimer and p65 homodimer, with binding to p50 homodimer being significantly weaker. Our results indicate that the nuclear localization s equence and the region C-terminal to it of the NF-kappa B p65 subunit is a major contributor to NF-kappa B I kappa B alpha complex formation. Addition ally, there are no contacts between the corresponding nuclear localization signal tetrapeptide of p50 and I kappa B alpha. A second set of interaction s involving the acidic C-terminal/PEST-like region of I kappa B alpha and t he NF-kappa B p65 subunit N-terminal domain also contributes binding energy toward formation of the complex. This interaction is highly dynamic and no nspecific in nature, as shown by oxidative cysteine cross-linking. Phosphor ylation of the C-terminal/ PEST-like region by casein kinase II further enh ances binding.