Regions of IkappaBalpha that are critical for its inhibition of NF-kappaB.DNA interaction fold upon binding to NF-kappaB

Abstract

Nuclear factor kappaB (NF-kappaB) transcription factors regulate genes responsible for critical cellular processes. IkappaBalpha, -beta, and -epsilon bind to NF-kappaBs and inhibit their transcriptional activity. The NF-kappaB-binding domains of IkappaBs contain six ankyrin repeats (ARs), which adopt a beta-hairpin/alpha-helix/loop/alpha-helix/loop architecture. IkappaBalpha appears compactly folded in the IkappaBalpha.NF-kappaB crystal structure, but biophysical studies suggested that IkappaBalpha might be flexible even when bound to NF-kappaB. Amide H/(2)H exchange in free IkappaBalpha suggests that ARs 2-4 are compact, but ARs 1, 5, and 6 are conformationally flexible. Amide H/(2)H exchange is one of few techniques able to experimentally identify regions that fold upon binding. Comparison of amide H/(2)H exchange in free and NF-kappaB-bound IkappaBalpha reveals that the beta-hairpins in ARs 5 and 6 fold upon binding to NF-kappaB, but AR 1 remains highly solvent accessible. These regions are implicated in various aspects of NF-kappaB regulation, such as controlling degradation of IkappaBalpha, enabling high-affinity interaction with different NF-kappaB dimers, and preventing NF-kappaB from binding to its target DNA. Thus, IkappaBalpha conformational flexibility and regions of IkappaBalpha folding upon binding to NF-kappaB are important attributes for its regulation of NF-kappaB transcriptional activity.

Fig. 1.

The crystal structure of IκBα (blue) bound to NF-κB (p50, green; p65, red) (22). Helix 3 and helix 4 in p65 (magenta), which flank its NLS, interact with ARs 1–3 in IκBα. The dimerization domains in both p50 and p65 form an extensive interface with IκBα ARs 3–6. The p50 and p65 dimerization domains and the p65 N-terminal domain contact the C-terminal PEST sequence of IκBα. The N-terminal domain of p50, not present in the structure, is not involved in IκBα binding (48, 49).

Fig. 2.

IκBα β-hairpin peptides exchange to different extents. (a) Pepsin cleavage of IκBα results in 25 peptides (bars below the sequence), which cover 74% of its sequence, but 6 peptides (dashed bars) can be analyzed only qualitatively. A schematic of an AR above the sequence shows the α-helices and β-sheets (23). Peptic peptides cover the β-hairpin region in all six ARs (gray bars). (b) A peptide that covers the β-hairpin region in AR 5 (m/z of MH⁺ = 2,165.08) becomes highly deuterated in 2 min (ii), seen as a large shift to higher mass compared with a nondeuterated control sample (i). (c) A peptide that covers the β-hairpin region in AR 3 (MH⁺ = 1,054.58) incorporates fewer deuterons in 2 min (ii), seen as a moderate shift to higher mass compared with a nondeuterated control (i).

Fig. 3.

Amide H/²H exchange in IκBα β-hairpins with and without NF-κB. (a) Deuterium incorporation in the β-hairpin in AR 1, MH⁺ = 1,761.85, shows only small differences in the extent of exchange in free (○) and NF-κB-bound (●) IκBα that may be due to protection at the IκBα·NF-κB interface. Deuterium incorporation in the β-hairpins in AR 2, MH⁺ = 1,679.87 (b), and AR 3, MH⁺ = 1054.58 (c), show no differences, and the β-hairpin in AR 4, MH⁺ = 1,221.57 (d), shows only a small change in the extent of exchange between free and NF-κB-bound IκBα. The β-hairpins in AR 5, MH⁺ = 2,165.08 (e), and AR 6, MH⁺ = 1,788.89 (f), show decreases in the extent of amide exchange in NF-κB-bound IκBα that are much larger than expected for protection at the IκBα·NF-κB interface. Error bars represent the standard deviation of triplicate reactions, and the y axis maximum corresponds to the total number of exchangeable amide protons in the peptide, except for f, which has only 13 amide protons. Insets show MALDI mass envelopes in nondeuterated controls (Top), free IκBα after 2 min of exchange (Middle), and NF-κB-bound IκBα after 2 min of exchange (Bottom).

Fig. 4.

Amide H/²H exchange in IκBα α-helices with and without NF-κB. (a) Deuterium incorporation in the α-helices of AR 4 shows no change between free (○) and NF-κB-bound (●) IκBα. (b) NF-κB (gray) does not contact the α-helices in IκBα AR 4 (cyan) in the IκBα·NF-κB crystal structure (22) (IκBα is shown in blue). (c) Deuterium incorporation in the α-helices of AR 6 shows a small decrease in the extent of exchange in NF-κB-bound IκBα, which may be due to protection at the IκBα·NF-κB interface. (d) NF-κB (gray) contacts the α-helices in IκBα AR 6 (cyan) in the crystal structure of the IκBα·NF-κB complex (22). Interacting residues are shown with ball-and-stick representation. Error bars and the y axis maximum are as in Fig. 3, except for c, which has only 15 amide protons.

Fig. 5.

IκBα AR 5 and 6 β-hairpins exchange less in NF-κB-bound IκBα. IκBα from the IκBα·NF-κB crystal structure (22) is colored according to percent exchange after 2 min in free IκBα (a) and NF-κB-bound IκBα (b) (NF-κB and regions of IκBα for which exchange is not reported are shown in gray). The AR 5 and 6 β-hairpins exchange much less in NF-κB-bound IκBα than in free IκBα. The extent of exchange of the β-hairpins in ARs 5 and 6 is similar to that in ARs 2–4 in the NF-κB-bound state.

Fig. 6.

The β-hairpins of IκBα ARs 5 and 6 are conformationally flexible only in free IκBα. (a) Deuterium incorporation after 2 min in NF-κB-bound IκBα in all regions (●) is highly correlated with the SASA of the corresponding region of IκBα, calculated from the IκBα·NF-κB crystal structure (22). The extent of amide exchange in the β-hairpins in ARs 5 and 6 (■) is plotted separately for contrast with b. The correlation coefficient remained 0.95 whether or not these data were included. Because the crystal structure lacks electron density for residues 66–69, a corrected exchange (see Materials and Methods) for residues 70–80 was correlated with its SASA. (b) Deuterium incorporation after 2 min in free IκBα (○) is well correlated with the SASA of the corresponding region of IκBα (see Materials and Methods), except for the AR 5 and 6 β-hairpins (□), which exchange much more than expected if free and NF-κB-bound IκBα had the same structure and dynamics. Error bars represent the standard deviation of triplicate exchange reactions and the deviation in SASA for the two complexes in Protein Data Bank ID 1NFI (22).