Mechanism of IκBα binding to NF-κB dimers

Abstract

X-ray crystal structures of the NF-κB-IκBα complex revealed an extensive and complex protein-protein interface involving independent structural elements present in both IκBα and NF-κB. In this study, we employ a gel electrophoretic mobility shift assay to assess and quantitate the relative contributions of the observed interactions toward overall complex binding affinity. IκBα 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 sequence and the region C-terminal to it of the NF-κB p65 subunit is a major contributor to NF-κB-IηBα complex formation. Additionally, there are no contacts between the corresponding nuclear localization signal tetrapeptide of p50 and IκBα. A second set of interactions involving the acidic C-terminal/PEST-like region of IκBα and the NF-κB p65 subunit N-terminal domain also contributes binding energy toward formation of the complex. This interaction is highly dynamic and nonspecific in nature, as shown by oxidative cysteine cross-linking. Phosphorylation of the C-terminal/PEST-like region by casein kinase II further enhances binding.