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Detection of a ternary complex of nf-kappa b and i kappa b alpha with DNA provides insights into how i kappa b alpha removes nf-kappa b from transcription sites

Academic Article
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Overview

authors

  • Sue, S. C.
  • Alverdi, V.
  • Komives, E. A.
  • Dyson, Jane

publication date

  • January 2011

journal

  • Proceedings of the National Academy of Sciences of the United States of America  Journal

abstract

  • It has been axiomatic in the field of NF-?B signaling that the formation of a stable complex between NF-?B and the ankyrin repeat protein I?B? precludes the interaction of NF-?B 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-?B-I?B? complex in solution. Stepwise addition of a DNA fragment containing the ?B binding sequence to the I?B?-NF-?B complex results in changes in the I?B? 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 I?B? from the complex. However, even at high concentrations of DNA, I?B? remains associated with NF-?B, indicated by the absence of resonances of the free N-terminal ankyrin repeats of I?B?. The I?B?-mediated release of NF-?B 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-?B to the well-structured N-terminal ankyrin repeats of I?B?. Subsequently the poorly folded C-terminal ankyrin repeats of I?B? would fold upon binding to the p50 and p65 dimerization domains of NF-?B, permitting the negatively charged C-terminal PEST sequence of I?B? to displace the bound DNA through a process of local mass action.
  • 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.

subject areas

  • Amino Acid Sequence
  • Animals
  • Ankyrin Repeat
  • Base Sequence
  • Binding Sites
  • Binding, Competitive
  • DNA
  • Humans
  • I-kappa B Proteins
  • Magnetic Resonance Spectroscopy
  • Models, Molecular
  • Multiprotein Complexes
  • NF-kappa B
  • NF-kappa B p50 Subunit
  • Protein Binding
  • Protein Multimerization
  • Protein Structure, Quaternary
  • Protein Structure, Tertiary
  • Transcription Factor RelA
  • Transcription, Genetic
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Research

keywords

  • signal transduction
  • transcription factor
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Identity

PubMed Central ID

  • PMC3029698

International Standard Serial Number (ISSN)

  • 0027-8424

Digital Object Identifier (DOI)

  • 10.1073/pnas.1014323108

PubMed ID

  • 21220295
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Additional Document Info

start page

  • 1367

end page

  • 1372

volume

  • 108

issue

  • 4

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