Ikappa Balpha functions through direct contacts with the nuclear localization signals and the DNA binding sequences of NF-kappaB.
We have determined the binding energies of complexes formed between Ikappa Balpha and the wild type and mutational variants of three different Rel/NF-kappaB 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-kappaB dimers and Ikappa Balpha, IkappaB alpha binds the NF-kappaB 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-kappaB 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 Ikappa Balpha. In the cell, Ikappa Balpha functions to retain NF-kappaB 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-kappaB in complex with Ikappa Balpha. A model of the interactions in the complex is proposed based on our observations and the crystal structures of Rel/NF-kappaB dimers and the ankyrin domains of related proteins.
Malek
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S
Department of Chemistry and Biochemistry, University of California, San Diego, California 92093-0359, USA.
Huxford
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T
Ghosh
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G
D001483:Base Sequence; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D019281:Dimerization; D016231:Fluorescence Polarization Immunoassay; D020672:I-kappa B Proteins; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism*; D019913:Nuclear Localization Signals; D011994:Recombinant Proteins / Q000378:metabolism
D017931:DNA Primers; D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D016328:NF-kappa B; D019913:Nuclear Localization Signals; D011994:Recombinant Proteins; D000072000:NF-KappaB Inhibitor alpha