<scp>NMR</scp> characterization of a 72 k<scp>D</scp>a transcription factor using differential isotopic labeling

Mukherjee, Sulakshana; Borin, Brendan N.; Quintas, Pedro O.; Dyson, H. Jane

doi:10.1002/pro.2853

Cited by 8 publications

(18 citation statements)

References 21 publications

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“…Here we characterize this ternary complex by NMR and cryoelectron microscopy and show that it is a feasible intermediate in the termination process. correspondence between the NMR spectrum of the full-length heterodimer and those of the component domains (9). The RelAp50 dimer is large for NMR studies (72 kDa), but we have been able to obtain NMR data, including resonance assignments, for this protein, using a combination of differential isotopic labeling and transverse relaxation-optimized spectroscopy (TROSY) (9).…”

Section: Resultsmentioning

confidence: 99%

“…correspondence between the NMR spectrum of the full-length heterodimer and those of the component domains (9). The RelAp50 dimer is large for NMR studies (72 kDa), but we have been able to obtain NMR data, including resonance assignments, for this protein, using a combination of differential isotopic labeling and transverse relaxation-optimized spectroscopy (TROSY) (9). We chose to determine assignments for only the RelA portion of the heterodimer, simplifying the NMR spectroscopy by perdeuteration of the p50 portion.…”

Section: Resultsmentioning

confidence: 99%

“…Resonance assignments for the NLS region of the RelA DD in complex with deuterated p50 DD were difficult to obtain, due to the disordered nature of this region in the absence of partners (9). The positions of key residues in the NLS, in both free and IκBα-bound forms, were confirmed by residue-specific labeling (9) and by comparison with the spectrum of a peptide with sequence corresponding to that of the NLS (10). The cross-peak of Y306 reports on the interaction of the NLS with IκBα.…”

Section: Resultsmentioning

confidence: 99%

“…Proteins were prepared as previously described (9,15). All proteins used for the NMR experiments [RelA RHR (residues 19-325), p50 RHR (37-363), IκBα (67-287)] were perdeuterated.…”

Section: Methodsmentioning

confidence: 99%

“…All of the 2D NMR spectra shown are clean-TROSY 1 H-15 N heteronuclear single quantum coherence (HSQC) spectra (17) obtained with nonuniform sampling (9,18), recorded on Bruker spectrometers at 800 MHz (with cryoprobe) or 900 MHz. One-dimensional Watergate experiments were used to obtain the DNA 1 H spectra.…”

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confidence: 99%

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

Mukherjee

Quintas

McNulty

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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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 ...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

“…Proteins were prepared as previously described (9,15). All proteins used for the NMR experiments [RelA RHR (residues 19-325), p50 RHR (37-363), IκBα (67-287)] were perdeuterated.…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

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

Mukherjee

Quintas

McNulty

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Comparison of backbone dynamics of the p50 dimerization domain of NFκB in the homodimeric transcription factor NFκB1 and in its heterodimeric complex with RelA (p65)

et al. 2019

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The nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFκB) transcription factors play a critical role in human immune response. The family includes homodimers and heterodimers of five component proteins, which mediate different transcriptional responses and bind preferentially to different DNA sequences. Crystal structures of DNA complexes show that the dimers of the Rel-homology regions are structurally very similar. Differing DNA sequence preference together with structural similarity suggests that the dimers may differ in their dynamics. In this study, we present the first near-complete 15 N, 13 C α/β , and H N backbone resonance assignments of two dimers of the dimerization domain (DD) of the NFκB1 (p50) protein (residues 241-351): the homodimer of two p50 domains and a heterodimer of the p50 DD with the p65 DD. As expected, the two dimers behave very similarly, with chemical shift differences between them largely concentrated in the dimer interface and attributable to specific differences in the amino acid sequences of p50 and p65. A comparison of the picosecond-nanosecond dynamics of the homo-and heterodimers also shows that the environment of p50 is similar, with an overall slightly reduced correlation time for the homodimer compared to the heterodimer, consistent with its slightly smaller molecular weight. These results demonstrate that NMR spectroscopy can be used to explore subtle changes in structure and dynamics that have the potential to give insights into differences in specificity that can be exploited in the design of new therapeutic agents. K E Y W O R D S{ 1 H} -15 N NOE, NFκB1, p50 dimerization domain, protein dynamics, R 1 /R 2 relaxation

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Domain Stability Regulated through the Dimer Interface Controls the Formation Kinetics of a Specific NF-κB Dimer

et al. 2021

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The NF-κB family of transcription factors is a key regulator of the immune response in the vertebrates. The family comprises five proteins that function as dimers formed in various combinations among the members, with the RelA−p50 dimer being physiologically the most abundant. While most of the 15 possible dimers are scarcely present in the cell with some remaining experimentally undetected to date, there are specific gene sets that are only activated by certain sparsely populated NF-κB dimers. The mechanism of transcription activation of such specific genes that are activated only by specific NF-κB dimers remains unclear. Here we show that the dimer interfacial residues control the stabilization of the global hydrogen bond network of the NF-κB dimerization domain, which, in turn, controls the thermodynamic stabilization of different NF-κB dimers. The relatively low thermodynamic stability of the RelA−RelA homodimer is critical as it facilitates the formation of the more stable RelA−p50 heterodimer. Through the modulation of the thermodynamic stability of the RelA−RelA homodimer, the kinetics of the RelA−p50 heterodimer formation can be regulated. This phenomenon provides an insight into the mechanism of RelA−RelA specific target gene regulation in physiology.

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NMR characterization of a 72 kDa transcription factor using differential isotopic labeling

Cited by 8 publications

References 21 publications

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

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

Comparison of backbone dynamics of the p50 dimerization domain of NFκB in the homodimeric transcription factor NFκB1 and in its heterodimeric complex with RelA (p65)

Domain Stability Regulated through the Dimer Interface Controls the Formation Kinetics of a Specific NF-κB Dimer

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