Mutation of Nonessential Cysteines Shows That the NF-κB Essential Modulator Forms a Constitutive Noncovalent Dimer That Binds IκB Kinase-β with High Affinity

Cote, Shaun M.; Gilmore, Thomas D.; Shaffer, Robert; Weber, Urs; Bollam, Rishitha; Golden, Mary S.; Glover, Kimberley; Herscovitch, Melanie; Ennis, Thomas; Allen, Karen N.; Whitty, Adrian

doi:10.1021/bi401368r

Cited by 21 publications

(110 citation statements)

References 59 publications

(291 reference statements)

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“…The data were fitted to a standard quadratic binding equation, as described in Materials and Methods, giving a best fit value for the binding affinity of FITC-IKKβ for NEMO of K D1 = 3.6 (95% CI = 2.4–5.0) nM ( n = 4), consistent with the best literature values for this interaction. 49,52,54,67 Figure 3C shows that binding of NEMO and FITC-IKKβ (each at 15 nM final concentration) is inhibited in a dose-dependent fashion by unlabeled IKKβ(701–745) synthetic peptide. The signal observed at maximum inhibition coincides with the minimum anisotropy value (A F ) measured in control wells containing FITC-IKKβ but no NEMO, confirming that a sufficient concentration of the unlabeled peptide inhibitor completely blocked the binding of FITC-IKKβ.…”

Section: Resultsmentioning

confidence: 99%

“…Recombinant NEMO was full-length protein (residues 2–419) containing a His6-tag to aid purification plus five cysteine-to-alanine substitutions, at positions 11, 76, 95, 131, and 167, which have been shown to improve the homogeneity and solubility of the protein without affecting IKKβ binding. 54 NEMO protein was expressed in E. coli and purified by Ni-NTA chromatography followed by gel filtration, as described. 54 …”

Section: Methodsmentioning

confidence: 99%

“…54 NEMO protein was expressed in E. coli and purified by Ni-NTA chromatography followed by gel filtration, as described. 54 …”

Section: Methodsmentioning

confidence: 99%

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Comprehensive Experimental and Computational Analysis of Binding Energy Hot Spots at the NF-κB Essential Modulator/IKKβ Protein–Protein Interface

et al. 2013

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We report a comprehensive analysis of binding energy hot spots at the protein-protein interaction (PPI) interface between NF-κB Essential Modulator (NEMO) and IκB kinase subunit β (IKKβ), an interaction that is critical for NF-κB pathway signaling, using experimental alanine scanning mutagenesis and also the FTMap method for computational fragment screening. The experimental results confirm that the previously identified NBD region of IKKβ contains the highest concentration of hot spot residues, the strongest of which are W739, W741 and L742 (ΔΔG = 4.3, 3.5 and 3.2 kcal/mol, respectively). The region occupied by these residues defines a potentially druggable binding site on NEMO that extends for ~16 Å to additionally include the regions that bind IKKβ L737 and F734. NBD residues D738 and S740 are also important for binding but do not make direct contact with NEMO, instead likely acting to stabilize the active conformation of surrounding residues. We additionally found two previously unknown hot spot regions centered on IKKβ residues L708/V709 and L719/I723. The computational approach successfully identified all three hot spot regions on IKKβ. Moreover, the method was able to accurately quantify the energetic importance of all hot spots residues involving direct contact with NEMO. Our results provide new information to guide the discovery of small molecule inhibitors that target the NEMO/IKKβ interaction. They additionally clarify the structural and energetic complementarity between “pocket-forming” and “pocket occupying” hot spot residues, and further validate computational fragment mapping as a method for identifying hot spots at PPI interfaces.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Comprehensive Experimental and Computational Analysis of Binding Energy Hot Spots at the NF-κB Essential Modulator/IKKβ Protein–Protein Interface

et al. 2013

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“…One common method to analyze NEMO mutant function is to re-express the NEMO protein in mouse NEMO knockout cells (Schröfelbauer et al, 2012;Cote et al, 2013). Such experiments have two limitations: 1) human NEMO proteins are being analyzed in mouse cells, and 2) to establish pure, selected populations of NEMO reconstituted cells can take a month or more.…”

Section: Discussionmentioning

confidence: 99%

CRISPR/Cas9-based Editing of a Sensitive Transcriptional Regulatory Element to Achieve Cell Type-Specific Knockdown of the NEMO Scaffold Protein

Babaei

Liu

Wuerzberger-Davis

et al. 2018

Preprint

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The use of alternative promoters for the cell type-specific expression of a given mRNA/protein is a common cell strategy. NEMO is a scaffold protein required for canonical NF-κB signaling. Transcription of the NEMO gene is primarily controlled by two promoters: one (promoter B) drives NEMO transcription in most cell types and the second (promoter A) is largely responsible for NEMO transcription in liver cells. Herein, we have used a CRISPR/Cas9-based approach to disrupt a core sequence element of promoter B, and this genetic editing essentially eliminates expression of NEMO mRNA and protein in 293T human kidney cells. By cell subcloning, we have isolated targeted 293T cell lines that express no detectable NEMO protein, have defined genomic alterations at promoter B, and do not support canonical NF-κB signaling in response to treatment with tumor necrosis factor (TNF). Nevertheless, non-canonical NF-κB signaling is intact in these NEMO-deficient cells. Expression of ectopic NEMO in the edited cells restores downstream NF-κB signaling in response to TNF. Targeting of the promoter B element does not substantially reduce NEMO expression (from promoter A) in the human SNU-423 liver cancer cell line. We have also used homology directed repair (HDR) to fix the promoter B element in a 293T cell clone. Overall, we have created a strategy for selectively eliminating cell type-specific expression from an alternative promoter and have generated 293T cell lines with a functional knockout of NEMO. The implications of these findings for further studies and for therapeutic approaches to target canonical NF-κB signaling are discussed.

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“…30 Fluorescence anisotropy was determined using a Tecan F500 Infinite plate reader, an excitation wavelength of 485 nm, and an emission wavelength of 525 nm as 26 …”

Section: Experimental Proceduresmentioning

confidence: 99%

Protein Engineering of the N-Terminus of NEMO: Structure Stabilization and Rescue of IKKβ Binding

et al. 2014

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NEMO is a scaffolding protein that, together with the catalytic subunits IKKα and IKKβ, plays an essential role in the formation of the IKK complex and in the activation of the canonical NF-κB pathway. Rational drug design targeting the IKK-binding site on NEMO would benefit from structural insight, but to date, the determination of the structure of unliganded NEMO has been hindered by protein size and conformational heterogeneity. Here we show how the utilization of a homodimeric coiled-coil adaptor sequence stabilizes the minimal IKK-binding domain NEMO(44–111) and furthers our understanding of the structural requirements for IKK binding. The engineered constructs incorporating the coiled coil at the N-terminus, C-terminus, or both ends of NEMO(44–111) present high thermal stability and cooperative melting and, most importantly, restore IKKβ binding affinity. We examined the consequences of structural content and stability by circular dichoism and nuclear magnetic resonance (NMR) and measured the binding affinity of each construct for IKKβ(701–745) in a fluorescence anisotropy binding assay, allowing us to correlate structural characteristics and stability to binding affinity. Our results provide a method for engineering short stable NEMO constructs to be suitable for structural characterization by NMR or X-ray crystallography. Meanwhile, the rescuing of the binding affinity implies that a preordered IKK-binding region of NEMO is compatible with IKK binding, and the conformational heterogeneity observed in NEMO(44–111) may be an artifact of the truncation.

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Mutation of Nonessential Cysteines Shows That the NF-κB Essential Modulator Forms a Constitutive Noncovalent Dimer That Binds IκB Kinase-β with High Affinity

Cited by 21 publications

References 59 publications

Comprehensive Experimental and Computational Analysis of Binding Energy Hot Spots at the NF-κB Essential Modulator/IKKβ Protein–Protein Interface

Comprehensive Experimental and Computational Analysis of Binding Energy Hot Spots at the NF-κB Essential Modulator/IKKβ Protein–Protein Interface

CRISPR/Cas9-based Editing of a Sensitive Transcriptional Regulatory Element to Achieve Cell Type-Specific Knockdown of the NEMO Scaffold Protein

Protein Engineering of the N-Terminus of NEMO: Structure Stabilization and Rescue of IKKβ Binding

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