Pre-folding IκBα alters control of NF-κB signaling

JOURNAL OF MOLECULAR BIOLOGY

卷:380

Article

Truhlar, Stephanie M. E.¹  Mathes, Erika¹  Cervantes, Caria F.¹  Ghosh, Gourisankar¹  Komives, Elizabeth A.¹ 

[1] Univ Calif San Diego, Dept Chem & Biochem, La Jolla, CA 92093 USA

关键词: coupled folding and binding;    transcription factor regulation;    protein-protein interactions;    ankyrin repeat;    ubiquitin-independent proteasome degradation;   

DOI  :  10.1016/j.jmb.2008.02.053

来源: Elsevier

【 摘 要 】

Transcription complex components frequently show coupled folding and binding but the functional significance of this mode of molecular recognition is unclear. I kappa B alpha binds to and inhibits the transcriptional activity of NF-kappa B via its ankyrin repeat (AR) domain. The beta-hairpins in ARs 5-6 in I kappa B alpha are weakly-folded in the free protein, and their folding is coupled to NF-kappa B binding. Here, we show that introduction of two stabilizing mutations in I kappa B alpha AR 6 causes ARs 5-6 to fold cooperatively to a conformation similar to that in NF-kappa B-bound I kappa B alpha. Free I kappa B alpha is degraded by a proteasome-dependent but ubiquitin-independent mechanism, and this process is slower for the pre-folded mutants both in vitro and in cells. Interestingly, the pre-folded mutants bind NF-kappa B more weakly, as shown by both surface plasmon resonance and isothermal titration calorimetry in vitro and immunoprecipitation experiments from cells. One consequence of the weaker binding is that resting cells containing these mutants show incomplete inhibition of NF-kappa B activation; they have significant amounts of nuclear NF-kappa B. Additionally, the weaker binding combined with the slower rate of degradation of the free protein results in reduced levels of nuclear NF-kappa B upon stimulation. These data demonstrate clearly that the coupled folding and binding of I kappa B alpha is critical for its precise control of NF-kappa B transcriptional activity. (C) 2008 Elsevier Ltd. All rights reserved.

【 授权许可】

Free