Binding of NFκB Appears to Twist the Ankyrin Repeat Domain of IκBα

Abstract

Total internal reflection fluorescence-based single-molecule Förster resonance energy transfer (FRET) measurements were previously carried out on the ankyrin repeat domain (ARD) of IκBα, the temporally regulated inhibitor of canonical NFκB signaling. Under native conditions, most of the IκBα molecules showed stable, high FRET signals consistent with distances between the fluorophores estimated from the crystal structures of the NFκB(RelA/p50)-IκBα complex. Similar high FRET efficiencies were found when the IκBα molecules were either free or in complex with NFκB(RelA/p50), and were interpreted as being consistent with the crystallographically observed ARD structure. An exception to this was observed when the donor and acceptor fluorophores were attached in AR3 (residue 166) and AR6 (residue 262). Surprisingly, the FRET efficiency was lower for the bound IκBα molecules (0.67) than for the free IκBα molecules (0.74), apparently indicating that binding of NFκB(RelA/p50) stretches the ARD of IκBα. Here, we conducted confocal-based single-molecule FRET studies to investigate this phenomenon in greater detail. The results not only recapitulated the apparent stretching of the ARD but also showed that the effect was more pronounced when the N-terminal domains (NTDs) of both RelA and p50 were present, even though the interface between NFκB(RelA/p50) and IκBα encompasses only the dimerization domains. We also performed mass spectrometry-detected amide hydrogen/deuterium exchange (HDXMS) experiments on IκBα as well as IκBα bound to dimerization-domain-only constructs or full-length NFκB(RelA/p50). Although we expected the stretched IκBα to have regions with increased exchange, instead the HDXMS experiments showed decreases in exchange in AR3 and AR6 that were more pronounced when the NFκB NTDs were present. Simulations of the interaction recapitulated the increased distance between residues 166 and 262, and also provide a plausible mechanism for a twisting of the IκBα ARD induced by interactions of the IκBα proline-glutamate-serine-threonine-rich sequence with positively charged residues in the RelA NTD.

DNA and IκBα Both Induce Long-Range Conformational Changes in NFκB

“…Although we analyzed 109 peptides for IκBα, we realized that a subset of 61 peptides achieve maximal coverage and information content. The results of the newer data, which include complete coverage of AR5, confirmed previous results and were recently published [12]. Similarly, although 69 peptides for RelA and 83 peptides for p50 were analyzed, a subset of 17 peptides from RelA and 18 peptides from p50 that achieve maximal coverage and information content are presented here (Supplementary Figure 1).…”

Functional importance of stripping in NFκB signaling revealed by a stripping-impaired IκBα mutant

“…Previous NMR experiments showed that resonances in AR3 broaden upon NFκB binding (20), and our results suggest DNA binding further weakens the AR3 structure. Previous single-molecule FRET studies suggested that AR3 senses events at the N-terminal domains of NFκB; in these studies, a subtle twisting of the AR domain accounted for the observed decrease in the FRET signal when the N-terminal domains were included in the binary complex and pointed to a slight structural rearrangement upon formation of the binary NFκB-IκBα complex (29). Here we see that when the ternary complex forms, AR3 senses what is bound to the NFκB N-terminal domains-in this case, DNA.…”

“…Murine N-terminal hexahistidine-RelA 19-321 /p50 39-350 heterodimer (NFκB) was coexpressed as described previously (20) and was purified by nickel affinity chromatography, cation exchange chromatography (Mono S; GE Healthcare), and size-exclusion chromatography (Superdex 200; GE Healthcare). Murine dimerization domain RelA with an N-terminal cysteine and dimerization domain p50 248-350 were expressed, purified, and prepared for surface plasmon resonance (SPR) as described (25,26,29,30). Immediately before the experiments, IκBα was purified by size-exclusion chromatography (Superdex 75; GE Healthcare), and NFκB and NFκB-IκBα complexes were purified by size-exclusion chromatography (Superdex 200) in 25 mM Tris (pH 7.5), 150 mM NaCl, 1 mM DTT, and 0.5 mM EDTA.…”

“…The peptides were identified from triplicate MS E analyses of 10 μM IκBα(WT), IκBα(5xPEST), or NFκB, and data were analyzed using PLGS 2.5 (Waters Corporation) as previously described (29). The peptides identified in PLGS then were analyzed in DynamX 3.0 (Waters Corporation) following previously published methods (29,34).…”

“…The peptides identified in PLGS then were analyzed in DynamX 3.0 (Waters Corporation) following previously published methods (29,34). Intracellular NFκB Nuclear Export Experiments.…”