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

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

Analysis of the RelA:CBP/p300 interaction reveals its involvement in NF-κB-driven transcription.

NF-κB plays a vital role in cellular immune and inflammatory response, survival, and proliferation by regulating the transcription of various genes involved in these processes. To activate transcription, RelA (a prominent NF-κB family member) interacts with transcriptional co-activators like CREB-binding protein (CBP) and its paralog p300 in addition to its cognate κB sites on the promoter/enhancer regions of DNA. The RelA:CBP/p300 complex is comprised of two components--first, DNA binding domain of RelA interacts with the KIX domain of CBP/p300, and second, the transcriptional activation domain (TAD) of RelA binds to the TAZ1 domain of CBP/p300. A phosphorylation event of a well-conserved RelA(Ser276) is prerequisite for the former interaction to occur and is considered a decisive factor for the overall RelA:CBP/p300 interaction. The role of the latter interaction in the transcription of RelA-activated genes remains unclear. Here we provide the solution structure of the latter component of the RelA:CBP complex by NMR spectroscopy. The structure reveals the folding of RelA-TA2 (a section of TAD) upon binding to TAZ1 through its well-conserved hydrophobic sites in a series of grooves on the TAZ1 surface. The structural analysis coupled with the mechanistic studies by mutational and isothermal calorimetric analyses allowed the design of RelA-mutants that selectively abrogated the two distinct components of the RelA:CBP/p300 interaction. Detailed studies of these RelA mutants using cell-based techniques, mathematical modeling, and genome-wide gene expression analysis showed that a major set of the RelA-activated genes, larger than previously believed, is affected by this interaction. We further show how the RelA:CBP/p300 interaction controls the nuclear response of NF-κB through the negative feedback loop of NF-κB pathway. Additionally, chromatin analyses of RelA target gene promoters showed constitutive recruitment of CBP/p300, thus indicating a possible role of CBP/p300 in recruitment of RelA to its target promoter sites

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

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

Super-Resolved Nuclear Magnetic Resonance Spectroscopy

We present a novel method that breaks the resolution barrier in nuclear magnetic resonance (NMR) spectroscopy, allowing one to accurately estimate the chemical shift values of highly overlapping or broadened peaks. This problem is routinely encountered in NMR when peaks have large linewidths due to rapidly decaying signals, hindering its application. We address this problem based on the notion of finite-rate-of-innovation (FRI) sampling, which is based on the premise that signals such as the NMR signal, can be accurately reconstructed using fewer measurements than that required by existing approaches. The FRI approach leads to super-resolution, beyond the limits of contemporary NMR techniques. Using this method, we could measure for the first time small changes in chemical shifts during the formation of a Gold nanorod-protein complex, facilitating the quantification of the strength of such interactions. The method thus opens up new possibilities for the application and acceleration of multidimensional NMR spectroscopy across a wide range of systems

Analysis of the RelA:CBP/p300 Interaction Reveals Its Involvement in NF-κB-Driven Transcription

<div><p>NF-κB plays a vital role in cellular immune and inflammatory response, survival, and proliferation by regulating the transcription of various genes involved in these processes. To activate transcription, RelA (a prominent NF-κB family member) interacts with transcriptional co-activators like CREB-binding protein (CBP) and its paralog p300 in addition to its cognate κB sites on the promoter/enhancer regions of DNA. The RelA:CBP/p300 complex is comprised of two components—first, DNA binding domain of RelA interacts with the KIX domain of CBP/p300, and second, the transcriptional activation domain (TAD) of RelA binds to the TAZ1 domain of CBP/p300. A phosphorylation event of a well-conserved RelA(Ser276) is prerequisite for the former interaction to occur and is considered a decisive factor for the overall RelA:CBP/p300 interaction. The role of the latter interaction in the transcription of RelA-activated genes remains unclear. Here we provide the solution structure of the latter component of the RelA:CBP complex by NMR spectroscopy. The structure reveals the folding of RelA–TA2 (a section of TAD) upon binding to TAZ1 through its well-conserved hydrophobic sites in a series of grooves on the TAZ1 surface. The structural analysis coupled with the mechanistic studies by mutational and isothermal calorimetric analyses allowed the design of RelA-mutants that selectively abrogated the two distinct components of the RelA:CBP/p300 interaction. Detailed studies of these RelA mutants using cell-based techniques, mathematical modeling, and genome-wide gene expression analysis showed that a major set of the RelA-activated genes, larger than previously believed, is affected by this interaction. We further show how the RelA:CBP/p300 interaction controls the nuclear response of NF-κB through the negative feedback loop of NF-κB pathway. Additionally, chromatin analyses of RelA target gene promoters showed constitutive recruitment of CBP/p300, thus indicating a possible role of CBP/p300 in recruitment of RelA to its target promoter sites.</p></div

Impaired RelA–TA2:CBP–TAZ1 interaction disrupts the negative feedback loop of the NF-κB pathway.

<p>(A) Expression of RelA mutants with respect to RelA(wt) in the RelA reconstituted <i>rela</i>⁻/⁻ cell lines. The protein expression levels of RelA(Ser276Ala) mutant was about three times lower than that for RelA(wt). For the quantitative estimation, the RelA to Actin signal ratio for each cell line was normalized with that for the RelA(wt) reconstituted <i>rela</i>⁻/⁻ cells (lower panel). (B) Model predictions for NF-κB (top) and total IκBα abundance (bottom) for different RelA expression levels (2×, 1.5×, 1×, 0.5×, and 0.2× relative to RelA(wt) values). In the nNF-κB curves, the peak is normalized to one for the highest level of nNF-κB activity and to zero for its levels in the resting cells (time = 0 min) for each individual expression level. Similarly, for the IκBα curves, the amount of IκBα in the resting cell (time = 0 min) is normalized to one and the minimum amount after degradation (basal levels) to zero following TNFα stimulation. (C) nRelA activity assay in response to TNFα stimulation as measured by EMSA using labeled κB probe and control NF-Y probe in the above-mentioned cells. The activity due to RelA binding to the κB probe was indicated by supershift of the EMSA band corresponding to the probe-bound NF-κB detected with anti-RelA antibody. 12 µg of NE was used for EMSA. (D) IκBα degradation and regeneration assay in RelA(wt/mutants) reconstituted <i>rela</i>⁻/⁻ cells following TNFα stimulation. IκBα protein levels after stimulation were monitored with respect to Actin. (E) Comparison of experimentally determined (symbols) and model predictions (solid line) based on adjusted RelA levels for NF-κB (left) and total IκBα abundance (right). Time courses for <i>rela</i>⁻/⁻ cells reconstituted with RelA(wt) (top), RelA(TA2) mutant (middle), and RelA(Ser276Ala) (bottom) stimulated with TNFα are shown. The experimental data were normalized as mentioned in panel (B) of this figure. The RMSD values correspond to the combined NF-κB and IκB datasets. (F) Comparison of experimentally determined (•) and model predictions (solid line) for NF-κB (left column) and total IκBα abundance (right column) for different degrees of suppression of IκBα mRNA production for RelA(TA2) mutant reconstituted <i>rela</i>⁻/⁻ cells.</p

NMR structure of the RelA–TA2:CBP–TAZ1 complex.

<p>RelA–TA2 is shown in green and TAZ1 in red. The three Zn²⁺ ligands are depicted in blue. The ordered region of the RelA fragment (Leu434–Val481) and TAZ1 (Ala345–Asp437) is depicted unless otherwise mentioned. (A) Twenty superimposed lowest energy NMR structures of RelA–TA2:CBP–TAZ1 complex. (B) Cartoon depiction of the lowest energy model of RelA–TA2:CBP–TAZ1 complex. (C) Electrostatic potential of solvent accessible surface of TAZ1 in complex with RelA–TA2. The positive potential is shown in blue and the negative in red. RelA–TA2 is shown as green ribbon with its acidic residues shown as red ball-and-sticks. RelA fragment (Gly430–Met483) is depicted in this figure.</p

Separating the components of the bipartite RelA:CBP interaction to delineate their individual roles.

<p>(A, Upper panel) GST-pulldown assay using in-vitro purified GST–KIX/GST–TAZ1 to pull down nRelA from NEs of wild-type 3T3 cells stimulated with TNFα for 30 min. EDTA was used to remove the Zn²⁺ from TAZ1, thereby disrupting the TAZ1 structure. (Lower panel) Coomassie-stained SDS-PAGE gel showing the inputs for the GST-tagged proteins. (B) GST-pulldown assay using in vitro purified GST–TAZ1 to pull down nRelA from the NEs of RelA(wt/mutants) reconstituted <i>rela</i>⁻/⁻ cells stimulated with TNFα for 30 min. RelA(Leu449Ala+Phe473Ala) and RelA(Leu449Ala+Phe473Ala+Ser467Ala) mutants have slower mobility on SDS-PAGE gels due to unknown reasons. The expression level of the RelA(Ser276Ala) mutant in the reconstituted cell line was lower than that for the RelA(wt) (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001647#pbio.1001647.s006" target="_blank">Figure S6</a>). Hence, a 3-fold excess of NEs was used for the RelA(Ser276Ala) mutant in this experiment. (C) Interaction of endogenous CBP with nRelA from the NEs of RelA(wt/mutants) reconstituted <i>rela</i>⁻/⁻ cells stimulated with TNFα for 30 min studied by co-immunoprecipitation assay of CBP/RelA followed by immunoblotting by RelA/CBP. The amount of NEs used for RelA(Ser276Ala) mutants was three times that of RelA(wt/TA2) mutants due to its lower expression levels. The RelA(Ser276Ala+Leu449Ala+Phe473Ala) mutant, which could potentially abolish the total RelA:CBP interaction, showed low and inconsistent expression and hence was not used in this study. * denotes IgG heavy chain. (D) The RelA mutants defective in RelA:CBP interaction also are defective binding to p300. The co-IP experiments for p300 was performed similarly to those in panel (C) of this figure. (E) RelA phosphorylated at Ser467 has higher CBP/p300 binding potential than the nonphosphorylated form. The immunoprecipitation assay was performed with α-RelA on CE (left column panels) and NE (right column panels) of <i>rela</i>⁻/⁻ cells reconstituted with RelA(wt) or RelA(Ser467Ala) mutant at three different time intervals after being stimulated with TNFα (5 ng/ml) in addition to the unstimulated cells. Co-immunoprecipitation assays were performed with α-CBP and α-p300 on NE in exactly the same manner as for the IP experiments above. The recruitment of nRelA by CBP/p300 is similar at 15 min relative to 30 min but diminishes at 45 min after TNFα stimulation despite the concentration of total RelA in the nucleus being significantly lower at 15 min after stimulation. Identical co-IP experiments with the RelA(Ser467Ala) mutant shows a direct proportionality in CBP/p300 binding with the concentrations of total nRelA at the different time points after stimulation. This indicates that the exclusively nuclear p-Ser467–RelA whose concentration peaks at about 10 to 15 min post-TNFα stimulation possesses a higher binding affinity for CBP/p300 compared to the nonphosphorylated form.</p