Stress-response transcription factors such as NFκB turn on hundreds of genes and must have a mechanism for rapid cessation of transcriptional activation. We recently showed that the inhibitor of NFκB signaling, IκBα, dramatically accelerates the dissociation of NFκB from transcription sites, a process we have called "stripping." To test the role of the IκBα C-terminal PEST (rich in proline, glutamic acid, serine, and threonine residues) sequence in NFκB stripping, a mutant IκBα was generated in which five acidic PEST residues were mutated to their neutral analogs. This IκBα(5xPEST) mutant was impaired in stripping NFκB from DNA and formed a more stable intermediate ternary complex than that formed from IκBα(WT) because DNA dissociated more slowly. NMR and amide hydrogen-deuterium exchange mass spectrometry showed that the IκBα(5xPEST) appears to be "caught in the act of stripping" because it is not yet completely in the folded and NFκB-bound state. When the mutant was introduced into cells, the rate of postinduction IκBα-mediated export of NFκB from the nucleus decreased markedly.transcription factor | binding kinetics | intrinsically disordered proteins | nuclear export | hydrogen-deuterium exchange S tress-response transcription factors turn on hundreds of genes, and their regulation requires robust activation as well as rapid and complete cessation of the ensuing response. A good example is the NFκB family of transcription factors, which responds to a large number of extracellular stress stimuli, including factors controlling inflammation and the immune response (1-3). Aberrant regulation of NFκB results in numerous disease states, including cancer (1, 4). The IκB family of inhibitors keeps NFκB in the cytoplasm (in the "off" state) (5). IκBα is the main temporally regulated IκB. When a stress signal is received, IκBα is degraded rapidly, releasing NFκB, which enters the nucleus, binds to κB DNA sites, and up-regulates gene expression (Fig. 1A). In a classic negative feedback loop, the promoter upstream of the IκBα gene is strongly up-regulated by NFκB. We previously showed that in vitro IκBα rapidly accelerates the dissociation of NFκB from many different DNA sequences containing the κB motif in a folding-upon-binding event (6, 7). Thus, removal of NFκB(RelA/p50) from its target sites is kinetically determined, a process we call "molecular stripping" (8). The kinetic control of transcription factor-DNA interactions represents a paradigm shift because these interactions typically are described with equilibrium-binding models (9, 10) and thus would require the formulation of novel models based on stochastic rates.For IκBα to strip NFκB from DNA, a ternary NFκB-DNA-IκBα complex must form at least transiently. A very transient NFκB-DNA-IκBα complex was indeed observed in stopped-flow fluorescence experiments (11). At high concentrations, signals corresponding to a ternary NFκB-DNA-IκBα complex were also observed by NMR (12, 13). Together the stopped-flow and NMR data showed that IκBα binds to the NFκB-DNA comple...

IκBα inhibits the transcription factor, NFκB, by forming a very tightly bound complex in which the ankyrin repeat domain (ARD) of IκBα interacts primarily with the dimerization domain of NFκB. The first four ankyrin repeats (ARs) of the IκBα ARD are well-folded, but the AR5-6 region is intrinsically disordered according to amide H/D exchange and protein folding/unfolding experiments. We previously showed that mutations towards the consensus sequence for stable ankyrin repeats resulted in a “prefolded” mutant. To investigate whether the consensus mutations were uniquely able to order the AR5-6 region, we used a predictor of protein disordered regions PONDR VL-XT to select mutations that would alter the intrinsic disorder towards a more ordered structure (D→O mutants). The algorithm predicted two mutations, E282W and P261F, neither of which correspond to the consensus sequence for ankyrin repeats. Amide exchange and CD were used to assess ordering. Although only the E282W was predicted to be more ordered by CD and amide exchange, stopped-flow fluorescence studies showed that both of the D→O mutants were less efficient at dissociating NFκB from DNA.