Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 108, Issue 4, Pages 1367-1372Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1014323108
Keywords
signal transduction; transcription factor
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Funding
- National Institutes of Health [GM71862]
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It has been axiomatic in the field of NF-kappa B signaling that the formation of a stable complex between NF-kappa B and the ankyrin repeat protein I kappa B alpha precludes the interaction of NF-kappa 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-kappa B-I kappa B alpha complex in solution. Stepwise addition of a DNA fragment containing the kappa B binding sequence to the I kappa B alpha-NF-kappa B complex results in changes in the I kappa B alpha 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 kappa B alpha from the complex. However, even at high concentrations of DNA, I kappa B alpha remains associated with NF-kappa B, indicated by the absence of resonances of the free N-terminal ankyrin repeats of I kappa B alpha. The I kappa B alpha-mediated release of NF-kappa 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-kappa B to the well-structured N-terminal ankyrin repeats of I kappa B alpha. Subsequently the poorly folded C-terminal ankyrin repeats of I kappa B alpha would fold upon binding to the p50 and p65 dimerization domains of NF-kappa B, permitting the negatively charged C-terminal PEST sequence of I kappa B alpha to displace the bound DNA through a process of local mass action.
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