(A) The crystal structure of IκBα (blue) bound to NF-κB (p50, green; p65, red; p65 nuclear localization sequence (NLS), magenta) 24. Residues mutated in this study, Y254, T257, C186, and A220, do not contact NF-κB, and they are depicted with ball-and-stick representation and colored cyan. The figure was prepared using PyMOL 74. (B) The sequences of the IκBα ankryin repeats (ARs) are aligned with the consensus sequence for a stable AR 29. Cyan triangles indicate residues mutated in this study. In the consensus sequence, black letters indicate highly conserved residues and gray letters indicate weaker conservation.

Equilibrium unfolding of WT and mutant IκBα using urea as a denaturant. The CD signal and the fluorescence of the single tryptophan in IκBα, W258, located in AR 6 (insets) were recorded simultaneously for urea titrations of various IκBα proteins. The cooperative CD unfolding transition shows that the Y254L (B), Y254L/T257A (D), and Y254L/T257A/C186P/A220P (E) mutants are slightly more stable than WT (A) IκBα, but the T257A (C) mutant has the same thermodynamic stability. Only mutants containing both Y254L and T257A (D and E) show cooperative unfolding transitions in the fluorescence of W258, which is located in AR 6 (insets).

Amide H/²H exchange in wild-type (black), Y254L/T257A (blue), and Y254L/T257A/C186P/A220P (green) IκBα. Deuterium incorporation in the β-hairpins in ARs 2 (A), 3 (B), and 4 (C) is similar in all three proteins; however, the β-hairpins in ARs 5 (D) and 6 (E) incorporate much less deuterium in the pre-folded mutants than in WT IκBα. The deuterium incorporation is normalized according to the number of backbone amides in the peptide. (F) The number of deuterons incorporated in each peptide in ARs 1–4 (closed circles) correlates extremely well with the calculated solvent accessible surface area (SASA) of the corresponding region of IκBα. The β-hairpins in ARs 5 and 6 (open circles) in free WT IκBα exchange to a much greater extent than predicted by their SASA (see cluster indicated by arrow), whereas the extent of exchange in these regions in the mutants are well correlated with their SASA. The average of three independent exchange experiments is reported, and the error bars represent the standard deviation of these experiments.

Y254L/T257A (blue) and Y254L/T257A/C186P/A220P (green) are degraded more slowly than WT IκBα (black) in vitro and in vivo. (A) Purified 20S proteasome was incubated with WT and mutant IκBα and the amount of protein remaining was detected by western blot (top) and quantified by densitometry measurements (bottom). (B) Stable cell-lines containing IκBα transgenes were treated with cycloheximide (CHX) to stop translation and the amount of protein remaining over time was detected by western blot (top). Densitometry quantification of two independent experiments is shown (bottom) with a combined fit of the data. (C) The C186P/A220P mutant (orange) is degraded faster than WT IκBα (black). An α-β-actin western blot, shown in panels B and C, shows the equivalent loading of all samples.

Y254L/T257A and Y254L/T257A/C186P/A220P bind more weakly than WT IκBα to NF-κB (p50_248–350/p65_190–321) in vitro and in vivo. (A) NF-κB (p50/p65 and p65/p65) was immunoprecipitated from lysates of stable cell-lines containing IκBα transgenes. Total IκBα (10% input samples) and NF-κB-bound IκBα (IP samples) levels were detected by western blot (top). The starting levels of IκBα are higher in the Y254L/T257A and Y254L/T257A/C186P/A220P mutants compared to WT IκBα, but much lower levels of NF-κB-bound IκBα are observed for both mutants compared to WT IκBα. The starting and immunoprecipitated levels of NF-κB (p65) are similar in all three cell-lines (bottom). There is no non-specific binding of IκBα or NF-κB to the protein G beads (beads alone samples). (B) ITC binding isotherm of NF-κB titrated into Y254L/T257A IκBα at 25 °C. Data were analyzed using a model for a single set of identical binding sites, and the observed K_D is 23 nM. (C–E) Surface plasmon resonance (Biacore) was used to determine the binding kinetics of NF-κB (immobilized via an N-terminal biotin tag on the p65 subunit) with (C) wild type IκBα (at concentrations of 1.55–59.7 nM), (D) Y254L/T257A IκBα (at concentrations of 6.89–118 nM) and (E) Y254L/T257A/C186P/A220P IκBα (at concentrations of 1.40–106 nM). The pre-folded mutants both dissociate much faster than WT. Data were analyzed using a 1:1 Langmuir binding model.

Cells containing pre-folded mutants show altered amounts of nuclear NF-κB compared to WT IκBα. (A) Nuclear NF-κB levels in resting cells, measured by EMSA, show an extremely small amount of nuclear NF-κB in cells containing WT IκBα, whereas a significant amount of nuclear NF-κB is seen in cells containing the pre-folded mutants, which is equivalent to the amount seen in cells deficient in IκBα (pBABE vector). (B) Schematic outlining stimulus-induced activation of NF-κB. IκBα binds to NF-κB and, in resting cells, this prevents its nuclear localization. However, the faster dissociation rates for the pre-folded mutants (gray arrow) result in a significant amount of free IκBα and unbound NF-κB, which can translocate into the nucleus. Furthermore, free IκBα basal degradation is slower in cells containing the pre-folded mutants (gray arrow), resulting in a further increase in free IκBα levels. Upon stimulation, NF-κB-bound IκBα is phosphorylated, which initiates rapid ubiquitination and degradation by the 26S proteasome. This releases NF-κB, which can then translocate into the nucleus, bind DNA, and activate transcription. (C) Measurement of the amount of phosphorylated IκBα after stimulation with TNF-α shows that the pre-folded mutants are phosphorylated at the same rate as WT IκBα. Since phosphorylation initiates signal-dependent degradation of NF-κB-bound IκBα, we expect that the pre-folded mutants will be degraded at the same rate as WT in response to stimulus, in contrast to the slower basal degradation rates of the free pre-folded mutants. (D) Upon stimulation with TNF-α, cells containing WT IκBα show a robust increase in nuclear NF-κB, measured by EMSA. Cells containing the pre-folded mutants also show an increase in nuclear NF-κB upon stimulation; however, the response is reduced compared to cells containing WT IκBα, but higher than that observed in cells deficient in IκBα (pBABE vector).