Structural characterization of the ternary complex that mediates termination of NF-κB signaling by IκBα

Abstract

The transcription factor NF-κB is used in many systems for the transduction of extracellular signals into the expression of signal-responsive genes. Published structural data explain the activation of NF-κB through degradation of its dedicated inhibitor IκBα, but the mechanism by which NF-κB-mediated signaling is turned off by its removal from the DNA in the presence of newly synthesized IκBα (termed stripping) is unknown. Previous kinetic studies showed that IκBα accelerates NF-κB dissociation from DNA, and a transient ternary complex between NF-κB, its cognate DNA sequence, and IκBα was observed. Here we structurally characterize the >100-kDa ternary complex by NMR and negative stain EM and show a modeled structure that is consistent with the measurements. These data provide a structural basis for previously unidentified insights into the molecular mechanism of stripping.transcriptional activation | NMR | negative stain electron microscopy | protein-DNA complex | protein-protein complex C ellular responses to extracellular signals depend on a complex array of protein-protein interactions, including signaling cascades within the cytoplasm involving different types of posttranslational modification, ultimately leading to the activation of factors that mediate transcription of the appropriate response genes. One of the best-understood signaling molecules is NF-κB, a family of inducible transcription factors that is present in almost all cell types in higher eukaryotes. Its activation through various stimuli elicits variable responses, leading to transcription of target genes necessary for the immune response, inflammation, cellular growth, differentiation, cell adhesion, and cell survival (1). In mammals, the NF-κB family comprises a set of homo-or heterodimers of five monomer units, termed RelA (also known as p65), RelB, c-Rel, p50, and p52. The most abundant and bestknown NF-κB is the heterodimer of RelA and p50, present in the cytoplasm of resting cells in complex with its inhibitor IκBα, which masks the nuclear localization signal (NLS) of RelA. Upon receipt of a stimulus, a signaling cascade results in the phosphorylation, ubiquitination, and degradation of the bound IκBα; the RelA NLS is released; and the NF-κB enters the nucleus. Signal-responsive genes are regulated by the binding of NF-κB to the appropriate promoter. The activated NF-κB response is terminated through various mechanisms, including resynthesis of IκBα; the corresponding gene, nfkbia, is a strongly induced target gene of NF-κB. Newly synthesized IκBα enters the nucleus, recombines with the DNA-bound NF-κB, and the complex reverts to the resting state in the cytoplasm. The newly synthesized IκBα acts as an important component of the negative feedback loop that is one of the mechanisms for termination of NF-κB activation (2).A number of studies have illuminated the structural basis for the protein-protein interactions that NF-κB undergoes during the activation process. The X-ray crystal structure of the complex of RelA-p50 with a cognate DNA ...

Oocyte exposure to ZnO nanoparticles inhibits early embryonic development through the γ-H2AX and NF-κB signaling pathways

“…In the current study, these phosphatases were determined, and it was found that PP4C, PP4R3β, PP4R2, PP2ACα, and PP2A were elevated by ZnO NPs in embryos (Figure 2D). Transcription factor NF-κB (nuclear factor-kappa B) is a critical regulator of multiple biological functions: cell growth, cell survival, innate and adaptive immunity, and others [101–104]. In the current study it was found that ZnO NPs treatment decreased NF-κB in embryos.…”

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

“…We previously showed that DNA binding could be monitored by observing the 1D 1 H spectrum of the distinctive imino resonances of the DNA (13). We used this technique to monitor the formation of the ternary NFκB-DNA-IκBα(5xPEST) complex and found that the complex was fully formed at a slight excess (1. chemical-shift perturbations, amide resonances also disappeared upon the formation of the ternary complex; the great majority of the missing peaks were in AR3 (Table S1).…”

“…Discussion IκBα(WT) efficiently strips NFκB from DNA (6,11). The mechanism of this process necessitates the formation of a ternary complex, which was extremely transient under stopped-flow conditions but could be observed under the high-concentration conditions of NMR experiments (11)(12)(13). Using coarse-grained simulations, we showed that at least part of the driving force for molecular stripping likely comes from electrostatic repulsion between the negatively charged IκBα PEST sequence and DNA (25).…”

“…A very transient NFκB-DNA-IκBα complex was indeed observed in stopped-flow fluorescence experiments (11). At high concentrations, signals corresponding to a ternary NFκB-DNA-IκBα complex were also observed by NMR (12,13). Together the stopped-flow and NMR data showed that IκBα binds to the NFκB-DNA complex so tightly that it effectively does not dissociate, leading to a complex in which the DNA is bound to the N-terminal domains of NFκB and IκBα is bound to the dimerization domains.…”

“…S3). Both kinetic experiments and NMR characterization of the ternary complex in the presence of excess DNA showed that IκBα does not dissociate from NFκB once it is bound (11)(12)(13)26). Here we show that in the stabilized ternary complex formed with the IκBα(5xPEST) mutant, chemical shifts for most IκBα residues in AR1-AR4 were unperturbed in the binary NFκB-IκBα and ternary NFκB-DNA-IκBα complexes.…”