Transfer of Flexibility between Ankyrin Repeats in IκBα upon Formation of the NF-κB Complex

The mechanism of inhibition of the transcriptional activator nuclear factor κB (NF-κB) by the inhibitor IκBα is central to the understanding of the control of transcriptional activity via this widely employed pathway. Previous studies suggested that IκBα, a modular protein with an NF-κB 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-κB than ANK 5 and 6. Here we report NMR studies that confirm the enhanced flexibility of ANK 5 and 6 in free IκBα. Upon binding of NF-κB, ANK 5 and 6 become well structured and rigid, but, somewhat surprisingly, other domains of the IκBα, 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 IκBα 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-κB 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 IκBα 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.