Biophysical characterization of the free IκBα ankyrin repeat domain in solution

Abstract

The crystal structure of IB␣ in complex with the transcription factor, nuclear factor -B (NF-B) shows six ankyrin repeats, which are all ordered. Electron density was not observed for most of the residues within the PEST sequence, although it is required for high-affinity binding. To characterize the folded state of IB␣ (67-317) when it is not in complex with NF-B, we have carried out circular dichroism (CD) spectroscopy, 8-anilino-1-napthalenesulphonic acid (ANS) binding, differential scanning calorimetry, and amide hydrogen/deuterium exchange experiments. The CD spectrum shows the presence of helical structure, consistent with other ankyrin repeat proteins. The large amount of ANS-binding and amide exchange suggest that the protein may have molten globule character. The amide exchange experiments show that the third ankyrin repeat is the most compact, the second and fourth repeats are somewhat less compact, and the first and sixth repeats are solvent exposed. The PEST extension is also highly solvent accessible. IB␣ unfolds with a T m of 42°C, and forms a soluble aggregate that sequesters helical and variable loop parts of the first, fourth, and sixth repeats and the PEST extension. The second and third repeats, which conform most closely to a consensus for stable ankyrin repeats, appear to remain outside of the aggregate. The ramifications of these observations for the biological function of IB␣ are discussed.

Repeat-protein folding: New insights into origins of cooperativity, stability, and topology

“…This type of conformational heterogeneity has been demonstrated elegantly for the six ankyrin repeats of IκBα. Hydrogen exchange studies show that ankyrin repeats 1, 5, and 6 are substantially disordered in the unbound state [59], but repeats 5 and 6 become structured on binding to NF-κB [60]. A similar disorder-order transition is seen crystallographically for the first repeat of the Notch ankyrin domain when binding the transcription factor CSL [51,61,62].…”

Mapping the Energy Landscape of Repeat Proteins using NMR-detected Hydrogen Exchange

“…Exactly analogous to our results, they found that central repeats are more protected from exchange than are repeats at both ends. 35 Figure 4 also shows the Ising model predictions for the denaturant-dependence of the protection factors for each helix. Overall, the model captures the behavior of the different helices supporting the idea that each helix is an independent folding unit.…”

Regions of IκBα that are critical for its inhibition of NF-κB·DNA interaction fold upon binding to NF-κB

“…1), I B␣ appears to be compactly folded (20,22). However, ARs 1, 5, and 6 in free I B␣ readily exchange most of their amide protons, indicating that they are highly solvent accessible and perhaps unfolded (35). Circular dichroism (CD) showed that all of the secondary structure was present in both free and NF-B-bound I B␣ (35).…”

“…However, ARs 1, 5, and 6 in free I B␣ readily exchange most of their amide protons, indicating that they are highly solvent accessible and perhaps unfolded (35). Circular dichroism (CD) showed that all of the secondary structure was present in both free and NF-B-bound I B␣ (35). However, CD does not probe tertiary structure or protein flexibility, both of which are probed by amide H/ 2 H exchange.…”

“…However, CD does not probe tertiary structure or protein flexibility, both of which are probed by amide H/ 2 H exchange. Binding of 8-anilino-1-naphthalenesulfonic acid (ANS) suggested that I B␣ may have highly dynamic, molten-globule-like regions, even in the NF-B-bound state (35).…”