title: Detection of a ternary complex of NF-kappaB and IkappaBalpha with DNA provides insights into how IkappaBalpha removes NF-kappaB from transcription sites.
abstract: It has been axiomatic in the field of NF-κB signaling that the formation of a stable complex between NF-κB and the ankyrin repeat protein IκBα precludes the interaction of NF-κB with DNA. Contradicting this assumption, we present stopped-flow fluorescence and NMR experiments that give unequivocal evidence for the presence of a ternary DNA-NF-κB-IκBα complex in solution. Stepwise addition of a DNA fragment containing the κB binding sequence to the IκBα-NF-κB complex results in changes in the IκBα NMR spectrum that are consistent with dissociation of the region rich in proline, glutamate, serine, and threonine (PEST) and C-terminal ankyrin repeat sequences of IκBα from the complex. However, even at high concentrations of DNA, IκBα remains associated with NF-κB, indicated by the absence of resonances of the free N-terminal ankyrin repeats of IκBα. The IκBα-mediated release of NF-κB from its DNA-bound state may be envisioned as the reverse of this process. The initial step would consist of the coupled folding and binding of the intrinsically disordered nuclear localization sequence of the p65 subunit of NF-κB to the well-structured N-terminal ankyrin repeats of IκBα. Subsequently the poorly folded C-terminal ankyrin repeats of IκBα would fold upon binding to the p50 and p65 dimerization domains of NF-κB, permitting the negatively charged C-terminal PEST sequence of IκBα to displace the bound DNA through a process of local mass action.
authors.affiliation: Department of Molecular Biology MB2, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037-1000, USA.
authors.forename: Shih-Che
authors.initials: SC
authors.lastname: Sue
authors.forename: Vera
authors.initials: V
authors.lastname: Alverdi
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Komives
authors.forename: H Jane
authors.initials: HJ
authors.lastname: Dyson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017089:Ankyrin Repeat / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D020672:I-kappa B Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000378:metabolism; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D052002:NF-kappa B p50 Subunit / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D051996:Transcription Factor RelA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics
chemical_list: D020672:I-kappa B Proteins; D046912:Multiprotein Complexes; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; C000606204:NFKBIA protein, human; D051996:Transcription Factor RelA; D000072000:NF-KappaB Inhibitor alpha; D004247:DNA