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 structural 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 quantitate the relative contributions of the observed interactions toward overall 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 sequence 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. Additionally, there are no contacts between the corresponding nuclear localization signal tetrapeptide of p50 and I kappa B alpha. A second set of interactions involving the acidic C-terminal/PEST-like region of I kappa B alpha and the NF-kappa B 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.
Phelps
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CB
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093, USA.
Sengchanthalangsy
L L
LL
Huxford
T
T
Ghosh
G
G
D001665:Binding Sites; D019281:Dimerization; D004926:Escherichia coli; D006801:Humans; D020672:I-kappa B Proteins / Q000737:chemistry* / Q000378:metabolism; D016328:NF-kappa B / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding
D020672:I-kappa B Proteins; D016328:NF-kappa B