Thermodynamics Reveal that Helix Four in the NLS of NF-κB p65 Anchors IκBα, Forming a Very Stable Complex

Department of Chemistry and Biochemistry, University of California-San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0378, USA.
Journal of Molecular Biology (Impact Factor: 4.33). 08/2006; 360(2):421-34. DOI: 10.1016/j.jmb.2006.05.014
Source: PubMed


IkappaBalpha is an ankyrin repeat protein that inhibits NF-kappaB transcriptional activity by sequestering NF-kappaB outside of the nucleus in resting cells. We have characterized the binding thermodynamics and kinetics of the IkappaBalpha ankyrin repeat domain to NF-kappaB(p50/p65) using surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). SPR data showed that the IkappaBalpha and NF-kappaB associate rapidly but dissociate very slowly, leading to an extremely stable complex with a K(D,obs) of approximately 40 pM at 37 degrees C. As reported previously, the amino-terminal DNA-binding domain of p65 contributes little to the overall binding affinity. Conversely, helix four of p65, which forms part of the nuclear localization sequence, was essential for high-affinity binding. This was surprising, given the small size of the binding interface formed by this part of p65. The NF-kappaB(p50/p65) heterodimer and p65 homodimer bound IkappaBalpha with almost indistinguishable thermodynamics, except that the NF-kappaB p65 homodimer was characterized by a more favorable DeltaH(obs) relative to the NF-kappaB(p50/p65) heterodimer. Both interactions were characterized by a large negative heat capacity change (DeltaC(P,obs)), approximately half of which was contributed by the p65 helix four that was necessary for tight binding. This could not be accounted for readily by the small loss of buried non-polar surface area and we hypothesize that the observed effect is due to additional folding of some regions of the complex.

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Available from: Carrie Croy, Nov 19, 2014
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    • "Free IκBα is rapidly degraded via a ubiquitin-independent pathway and its halflife is less than 10 min. Binding to NFκB results in a high-affinity (picomolar) complex and increases the IκBα half-life to many hours [80]. These interactions inhibit nuclear localization of NFκB and its transcriptional activity. "
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    ABSTRACT: Specific molecular recognition is assumed to require a well-defined set of contacts and devoid of conformational and interaction ambiguities. Growing experimental evidence demonstrates however, that structural multiplicity or dynamic disorder can be retained in protein complexes, termed as fuzziness. Fuzzy regions establish alternative contacts between specific partners usually via transient interactions. Nature often tailors the dynamic properties of these segments via post-translational modifications or alternative splicing to fine-tune affinity. Most experimentally characterized fuzzy complexes are involved in regulation of gene-expression, signal transduction and cell-cycle regulation. Fuzziness is also characteristic to viral protein complexes, cytoskeleton structure, and surprisingly in a few metabolic enzymes. A plausible role of fuzzy complexes in increasing half-life of intrinsically disordered proteins is also discussed. Copyright © 2015. Published by Elsevier B.V.
    FEBS letters 07/2015; 589(19). DOI:10.1016/j.febslet.2015.07.022 · 3.17 Impact Factor
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    • "However, there was no evidence of p50 knockdown after siRNA treatment. Previous studies have shown that the NF-jB homo-and heterodimer form a very stable complex, with the half-life of IjBa in resting cells estimated at longer than 48 h [45] [46]. Regardless, this did not appear to affect the abrogation of TNF-a mediated suppression of PPARa, suggesting that a p50/p65 heterodimer could be a requirement for TNF-a mediated suppression of PPARa. "
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    ABSTRACT: Peroxisome proliferator activated receptor-alpha (PPARα) plays a major role in the regulation of lipid and glucose homeostasis, and inflammatory responses. The objectives of the study were to systematically investigate the effects of TNF-α and its regulatory pathway on PPARα expression in HepG2 cells using Real-Time RT-PCR and western blot analysis. Here, TNF-α suppressed PPARα mRNA expression in a dose- and time-dependent manner at the level of gene transcription. Pre-treatment of cells with 10μM of Wedelolactone for 2h was sufficient to restore PPARα expression to basal levels and also affected the expression of PPARα-regulated genes. This study also demonstrated that TNF-α represses PPARα expression by augmenting the activity of canonical NF-κB signalling pathway. This was shown by the abrogation of TNF-α-mediated PPARα down-regulation, after both p65 and p50 were knocked down via siRNA. The IKK contributes to IκBα degradation and mediates inducible phosphorylation of p105 at Ser933. Surprisingly, phosphorylation of p65 at Ser468 and Ser536 were severely abrogated with Wedelolactone inhibition, suggesting that Ser468 and Ser536, but not Ser276, may mediate the TNF-α inhibitory action on PPARα gene expression. These results suggest that TNF-α might, at least in part, suppress PPARα expression through activation of IKK/p50/p105/p65 pathway. Furthermore, phosphorylation of p65 at Ser468 and Ser536 may play a crucial role in the mechanism that limits PPARα production in the human HepG2 cells.
    Cytokine 01/2013; 61(1):266-274. DOI:10.1016/j.cyto.2012.10.007 · 2.66 Impact Factor
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    • "The binding interface between protein 4.1 and the erythrocyte anion exchanger is an example of hydrophobic side chains flanked by charged residues that are important for binding (Jöns and Drenckhahn 1992). Hydrophobic as well as electrostatic interactions participate in high affinity binding of spectrin and ankyrin (Kolondra et al. 2010) and of NFκB and IκBα (Bergqvist et al. 2006), which we used to model the high affinity binding site of obscurin (Busby et al. 2010). Consistent with these earlier studies, we show here that hydrophobic interactions contribute significantly to the binding of obscurin to the ALRs of sAnk1. "
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    ABSTRACT: Abstract Small ankyrin-1 is a splice variant of the ANK1 gene that binds to obscurin A. Previous studies have identified electrostatic interactions that contribute to this interaction. In addition, molecular dynamics (MD) simulations predict four hydrophobic residues in a 'hot spot' on the surface of the ankyrin-like repeats of sAnk1, near the charged residues involved in binding. We used site-directed mutagenesis, blot overlays and surface plasmon resonance assays to study the contribution of the hydrophobic residues, V70, F71, I102 and I103, to two different 30-mers of obscurin that bind sAnk1, Obsc₆₃₁₆₋₆₃₄₅ and Obsc₆₂₃₁₋₆₂₆₀. Alanine mutations of each of the hydrophobic residues disrupted binding to the high affinity binding site, Obsc₆₃₁₆₋₆₃₄₅. In contrast, V70A and I102A mutations had no effect on binding to the lower affinity site, Obsc₆₂₃₁₋₆₂₆₀. Alanine mutagenesis of the five hydrophobic residues present in Obsc₆₃₁₆₋₆₃₄₅ showed that V6328, I6332, and V6334 were critical to sAnk1 binding. Individual alanine mutants of the six hydrophobic residues of Obsc₆₂₃₁₋₆₂₆₀ had no effect on binding to sAnk1, although a triple alanine mutant of residues V6233/I6234/I6235 decreased binding. We also examined a model of the Obsc₆₃₁₆₋₆₃₄₅-sAnk1 complex in MD simulations and found I102 of sAnk1 to be within 2.2Å of V6334 of Obsc₆₃₁₆₋₆₃₄₅. In contrast to the I102A mutation, mutating I102 of sAnk1 to other hydrophobic amino acids such as phenylalanine or leucine did not disrupt binding to obscurin. Our results suggest that hydrophobic interactions contribute to the higher affinity of Obsc₆₃₁₆₋₆₃₄₅ for sAnk1 and to the dominant role exhibited by this sequence in binding.
    Molecular Membrane Biology 03/2012; 29(2):36-51. DOI:10.3109/09687688.2012.660709 · 1.69 Impact Factor
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