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...

We recently discovered that IκBα enhances the rate of release of NFκB from DNA target sites in a process we have termed molecular stripping. Coarse-grained molecular dynamics simulations of the stripping pathway revealed two mechanisms for the enhanced release rate; the negatively charged PEST region of IκBα electrostatically repels the DNA, and binding of IκBα appears to twist the NFκB heterodimer so that DNA can no longer bind. Here we report amide hydrogen/deuterium exchange data that reveals long-range allosteric changes in the NFκB (RelA-p50) heterodimer induced by DNA or IκBα binding. The data suggest that the two immunoglobulin-like subdomains of each Rel-homology region, which are connected by a flexible linker in the heterodimer, communicate in such a way that when DNA binds to the N-terminal DNA binding domains, the nuclear localization signal becomes more highly exchanging. Conversely, when IκBα binds to the dimerization domains, amide exchange throughout the DNA binding domains is decreased as if the entire domain is becoming globally stabilized. The results help understand how the subtle mechanism of molecular stripping actually occurs.

Previous single-molecule fluorescence resonance energy transfer (smFRET) studies in which the second and sixth ankyrin repeats (ARs) of IκBα were labeled with FRET pairs showed slow fluctuations as if the IκBα AR domain was unfolding in its native state. To systematically probe where these slow dynamic fluctuations occur, we now present data from smFRET studies wherein FRET labels were placed at ARs 1 and 4 (mutant named AR 1–4), at ARs 2 and 5 (AR 2–5), and at ARs 3 and 6 (AR 3–6). The results presented here reveal that AR 6 most readily detaches/unfolds from the AR domain, undergoing substantial fluctuations at room temperature. AR 6 has fewer stabilizing consensus residues than the other IκBα ARs, probably contributing to the ease with which AR 6 “loses grip”. AR 5 shows almost no fluctuations at room temperature, but a significant fraction of molecules shows fluctuations at 37 °C. Introduction of stabilizing mutations that are known to fold AR 6 dampen the fluctuations of AR 5, indicating that the AR 5 fluctuations are likely due to weakened inter-repeat stabilization from AR 6. AR 1 also fluctuates somewhat at room temperature, suggesting that fluctuations are a general behavior of ARs at ends of AR domains. Remarkably, AR 1 still fluctuates in the bound state, but mainly between 0.6 and 0.9 FRET efficiency, whereas in the free IκBα, the fluctuations extend to <0.5 FRET efficiency. Overall, our results provide a more complete picture of the energy landscape of the native state dynamics of an AR domain.

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.

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