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Transfer of flexibility between ankyrin repeats in IkappaB* upon formation of the NF-kappaB complex.

Abstract：

:The mechanism of inhibition of the transcriptional activator nuclear factor kappaB (NF-kappaB) by the inhibitor IkappaB* is central to the understanding of the control of transcriptional activity via this widely employed pathway. Previous studies suggested that IkappaB* , a modular protein with an NF-kappaB binding domain consisting of six ankyrin repeat domains (ANKs), shows differential flexibility, with ANK 1-4 apparently more rigid in solution in the absence of NF-kappaB than ANK 5 and 6. Here we report NMR studies that confirm the enhanced flexibility of ANK 5 and 6 in free IkappaB* . Upon binding of NF-kappaB, ANK 5 and 6 become well structured and rigid, but, somewhat surprisingly, other domains of the IkappaB* , which were relatively rigid in the free protein, become significantly more flexible. Due to the high molecular masses of the component proteins and the complexes, we employ a hierarchical experimental plan to maximize the available information on local flexibility in the ankyrin repeat domains. Backbone resonances of the 221-residue IkappaB* protein were assigned firstly in a smaller construct consisting of ankyrin repeats 1-4. These assignments could be readily transferred to the spectra of the construct containing six repeats, both free and complexed with various combinations of the NF-kappaB p50 and p65 domains. Transverse relaxation optimized spectroscopy-type NMR experiments on differentially labeled proteins enabled information on backbone structure and dynamics to be obtained, even in complexes with molecular masses approaching 100 kDa. Changes in the flexibility and stability of the various ankyrin repeat domains of IkappaB* complex formation take a variety of forms depending on the position of the domain in the complex, providing a variety of examples of the structural and functional utility of intrinsically unstructured or partly folded protein domains.

journal_name

J Mol Biol

journal_title

Journal of molecular biology

authors

Sue SC,Cervantes C,Komives EA,Dyson HJ

doi

10.1016/j.jmb.2008.05.048

subject

Has Abstract

pub_date

2008-07-25 00:00:00

pages

917-31

issue

eissn

0022-2836

issn

1089-8638

pii

S0022-2836(08)00629-3

journal_volume

380

pub_type

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Transfer of flexibility between ankyrin repeats in IkappaB* upon formation of the NF-kappaB complex.