Predicted disorder-to-order transition mutations in IκBα disrupt function

Abstract

IκBα inhibits the transcription factor, NFκB, by forming a very tightly bound complex in which the ankyrin repeat domain (ARD) of IκBα interacts primarily with the dimerization domain of NFκB. The first four ankyrin repeats (ARs) of the IκBα ARD are well-folded, but the AR5-6 region is intrinsically disordered according to amide H/D exchange and protein folding/unfolding experiments. We previously showed that mutations towards the consensus sequence for stable ankyrin repeats resulted in a “prefolded” mutant. To investigate whether the consensus mutations were uniquely able to order the AR5-6 region, we used a predictor of protein disordered regions PONDR VL-XT to select mutations that would alter the intrinsic disorder towards a more ordered structure (D→O mutants). The algorithm predicted two mutations, E282W and P261F, neither of which correspond to the consensus sequence for ankyrin repeats. Amide exchange and CD were used to assess ordering. Although only the E282W was predicted to be more ordered by CD and amide exchange, stopped-flow fluorescence studies showed that both of the D→O mutants were less efficient at dissociating NFκB from DNA.

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

“…Human IκBα 67-287 (IκBα) mutants were produced using site-directed mutagenesis, and the proteins were expressed and purified as described (30 (31). 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).…”

“…Sensorgrams were recorded on a GE Biacore 3000 instrument using streptavidin chips (GE Healthcare) by immobilizing 150, 250, and 350 response units of p50 dd /biotin-RelA dd on flow cells 2, 3, and 4, respectively, leaving flow cell 1 unmodified for reference subtraction. NFκB-binding experiments were conducted on IκBα(WT) and IκBα(5xPEST), and the data were analyzed as described (18,30).…”

“…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.…”

Telomerase and Cancer Research

“…We observe that in both the cases, the designed proteins and the Toll-like receptor 3, the regions where the wedges are located have a higher proportion of highly frustrated interactions. We have shown for the ANK protein family, that insertions within and in between adjacent repeats are enriched in this type of energetic conflicts [26], most likely because of functional constraints like surface adaptation to bind the target, as suggested in the case of LRRs or for dynamic and regulatory reasons like in the IκBα case [29,33].…”

Quarterly intrinsic disorder digest (April–May–June, 2014)

“…Dembinski et al investigated mutations that promote disorder-to-order transitions in IkBa and showed that these mutations decreased the efficiency of IkBa in inhibiting NFkB. 39 Regulation is a key function of IDPRs. JaramilloTatis et al developed a new SUMO-expression and purification protocol for the preparation of a full length murine SCP1/GIP, and were able to observe the interaction between murine SCP1/GIP and the intrinsically disordered BG21.…”