Functional dynamics of the folded ankyrin repeats of I kappa B alpha revealed by nuclear magnetic resonance

Abstract

Inhibition of nuclear factor kappaB (NF-kappaB) is mainly accomplished by IkappaB alpha, which consists of a signal response sequence at the N-terminus, a six-ankyrin repeat domain (ARD) that binds NF-kappaB, and a C-terminal PEST sequence. Previous studies with the ARD revealed that the fifth and sixth repeats are only partially folded in the absence of NF-kappaB. Here we report NMR studies of a truncated version of IkappaB alpha, containing only the first four ankyrin repeats, IkappaB alpha(67-206). This four-repeat segment is well-structured in the free state, enabling full resonance assignments to be made. H-D exchange, backbone dynamics, and residual dipolar coupling (RDC) experiments reveal regions of flexibility. In addition, regions consistent with the presence of micro- to millisecond motions occur periodically throughout the repeat structure. Comparison of the RDCs with the crystal structure gave only moderate agreement, but an ensemble of structures generated by accelerated molecular dynamics gave much better agreement with the measured RDCs. The regions showing flexibility correspond to those implicated in entropic compensation for the loss of flexibility in ankyrin repeats 5 and 6 upon binding to NF-kappaB. The regions showing micro- to millisecond motions in the free protein are the ends of the beta-hairpins that directly interact with NF-kappaB in the complex.

Figure 1

Sequence alignment between individual repeats of the IκBα AR, with secondary structural elements shown schematically below the sequence. The last line corresponds to the consensus for a stable ankyrin repeat. Residues that correspond to the consensus in each repeat are shown in bold. The IκBα(67−206) fragment is denoted with brackets.

Figure 2

800 MHz ¹H−¹⁵N TROSY-HSQC spectrum of [²H,¹⁵N,¹³C]IκBα(67−206). The protein concentration was 0.5 mM in 25 mM Tris (pH 7.5), 50 mM NaCl, 50 mM arginine, 50 mM glutamic acid, 5 mM CHAPS, 1 mM EDTA, 1 mM dithiothreitol (DTT), and 2 mM NaN₃ in 90% H₂O and 10% D₂O at 293 K. Assignments for the backbone amides are labeled.

Figure 3

Model-free parameters calculated from the ¹⁵N relaxation data of free IκBα(67−206) using TENSOR2. (A) Generalized order parameters (S²) of N−H vectors plotted as a function of residue number. Results from anisotropic and isotropic models were similar. (B) Apparent chemical and conformational exchange contribution (R_ex) to the transverse relaxation rate R₂. Residues exhibiting R_ex values are located in areas of decreased S² values. Results from isotropic (●) and anisotropic (○) fits are shown. Secondary structure elements determined from the crystal structure of the IκBα·NF-κB complex are shown schematically at the top for the sake of comparison.

Figure 4

Experimentally measured ¹H−¹⁵N residual dipolar couplings of IκBα(67−206) plotted as a function of residue number. Secondary structure elements determined from the crystal structure of the IκBα·NF-κB complex are shown schematically at the top for the sake of comparison. Helices show consecutive negative RDC values of similar magnitudes, as expected for straight structural arrays, such as α-helices in which N−H bonds are aligned parallel with the helix axis and retain the same orientation with respect to the reference frame. The similar ranges of RDC values for individual helices suggest that the α-helices of free IκBα(67−206) in solution are oriented similarly with respect to the alignment tensor.

Figure 5

(A) Plot of observed vs theoretical residual dipolar couplings measured with PALES for IκBα(67−206) (●) and SVD (○) using the crystal structure of the IκBα·NF-κB complex [Protein Data Bank entry 1IKN(15)]. (B) Plot of observed vs AMD-calculated residual dipolar couplings for IκBα(67−206). There is significant improvement in the correlation of the AMD-calculated RDCs with the observed RDCs compared to the results from PALES. For the RDC measurements, an aligned solution was prepared by adding bacteriophage Pf1 (Asla Biotech) to a [¹⁵N]IκBα(67−206) sample. All spectra were acquired at 293 K.

Figure 6

(A) Structure of IκBα(67−206) from the IκBα·NF-κB complex [Protein Data Bank entry 1NFI(16)] showing the spin relaxation order parameters (S²) determined from the TENSOR2 analysis of the R₁, R₂, and hNOE data. (B) Ensemble of structures from the AMD simulation using the optimal torsional acceleration level for the best reproduction of the experimental RDCs: E_b(dih) − V(dih) = 600 kcal/mol, and α(dih) = 120 kcal/mol. The calculated order parameters (S²) determined from the N−H bond vectors from the ensemble-weighted average are shown on the structures. The color scales for panels A and B are from red to blue for S² values from 0.1 to 1.0, respectively. (C) Plot of the experimental order parameters (as in Figure 3) (●) compared to those calculated from the optimized AMD simulation (○).

Figure 7

Plot of the experimentally determined order parameters (as in Figure 3) for the free protein (●) compared with those for NF-κB-bound IκBα from previously published data (39) (○).

Figure 8

Structure of the IκBα·NF-κB complex [Protein Data Bank entry 1NFI (16)] showing those residues of IκBα(67−206) with significant R_ex colored red.