Mutation of Non-Essential Cysteines Shows that NF-κB Essential Modulator (NEMO) Forms a Constitutive Noncovalent Dimer that Binds IκB Kinase-β (IKKβ) with High Affinity.

Biochemistry (Impact Factor: 3.02). 11/2013; 52(51). DOI: 10.1021/bi401368r
Source: PubMed


NEMO (NF-κB essential modulator) associates with the catalytic subunits IKKα and IKKβ to form the IκB kinase (IKK) complex, and is a key regulator of NF-κB pathway signaling. Biochemical and structural characterization of NEMO has been challenging, however, leading to conflicting data on basic biochemical properties such as the oligomeric state of active NEMO and its binding affinity for IKKβ. We show that up to seven of NEMO's 11 cysteine residues can be mutated to generate recombinant full-length NEMO that is highly soluble and active. Using a fluorescence anisotropy binding assay we show that full-length NEMO binds a 44-mer peptide encompassing residues 701-745 of IKKβ with KD = 2.2 ± 0.8 nM. The IKKβ binding affinities of mutants with five and seven Cys-to-Ala substitutions are indistinguishable from that of wild-type NEMO. Moreover, when expressed in NEMO /- fibroblasts, the 5xAla and 7xAla NEMO mutants can interact with cellular IKKβ and restore NF κB signaling to provide protection against TNFα-induced cell death. Treatment of the NEMO-reconstituted cells with H2O2 led to formation of covalent dimers for wild-type NEMO and the 5xAla mutant, but not for the 7xAla mutant, confirming that Cys54 and/or Cys347 can mediate inter-chain disulfide bonding. However, the IKKβ binding affinity of NEMO is unaffected by the presence or absence of inter-chain disulfide bonding at Cys54 - which lies within the IKKβ binding domain of NEMO - or at Cys347, indicating that NEMO exists as a noncovalent dimer independent of the redox state of its cysteines. This conclusion was corroborated by the observation that the secondary structure content of the NEMO, its thermal stability, and its activity in cells were independent of the presence or absence of inter-chain disulfide bonds.

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    ABSTRACT: 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-ĸB pathway. Rational drug design targeting the IKK binding site on NEMO would benefit from structural insight, but to date the structure determination 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 IKKß binding affinity. We examined the consequences on structural content and stability by circular dichoism and nuclear magnetic resonance and measured 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 to engineer 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 pre-ordered 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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    Preview · Article · Nov 2014 · Biochemistry
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    ABSTRACT: NF-κB essential modulator, NEMO, plays a key role in canonical NF-κB signaling induced by a variety of stimuli, including cytokines and genotoxic agents. To dissect the different biochemical and functional roles of NEMO in NF-κB signaling, various mutant forms of NEMO have been previously analyzed. However, transient or stable overexpression of wild-type NEMO can significantly inhibit NF-κB activation, thereby confounding the analysis of NEMO mutant phenotypes. What levels of NEMO overexpression lead to such an artifact and what levels are tolerated with no significant impact on NEMO function in NF-κB activation are currently unknown. Here we purified full-length recombinant human NEMO protein and used it as a standard to quantify the average number of NEMO molecules per cell in a 1.3E2 NEMO-deficient murine pre-B cell clone stably reconstituted with full-length human NEMO (C5). We determined that the C5 cell clone has an average of 4 x 105 molecules of NEMO per cell. Stable reconstitution of 1.3E2 cells with different numbers of NEMO molecules per cell has demonstrated that a 10-fold range of NEMO expression (0.6-6x105 molecules per cell) yields statistically equivalent NF-κB activation in response to the DNA damaging agent etoposide. Using the C5 cell line, we also quantified the number of NEMO molecules per cell in several commonly employed human cell lines. These results establish baseline numbers of endogenous NEMO per cell and highlight surprisingly normal functionality of NEMO in the DNA damage pathway over a wide range of expression levels that can provide a guideline for future NEMO reconstitution studies.
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