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Biochemistry ; 52(51): 9141-54, 2013 Dec 23.
Article in English | MEDLINE | ID: mdl-24266532

ABSTRACT

NEMO (NF-κB essential modulator) associates with catalytic subunits IKKα and IKKß 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 about basic biochemical properties such as the oligomeric state of active NEMO and its binding affinity for IKKß. 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 IKKß with a K(D) of 2.2 ± 0.8 nM. The IKKß 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 five-Ala and seven-Ala NEMO mutants can interact with cellular IKKß and restore NF-κB signaling to provide protection against tumor necrosis factor α-induced cell death. Treatment of the NEMO-reconstituted cells with H2O2 led to the formation of covalent dimers for wild-type NEMO and the five-Ala mutant, but not for the seven-Ala mutant, confirming that Cys54 and/or Cys347 can mediate interchain disulfide bonding. However, the IKKß binding affinity of NEMO is unaffected by the presence or absence of interchain disulfide bonding at Cys54, which lies within the IKKß 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 NEMO and its thermal stability were independent of the presence or absence of interchain disulfide bonds.


Subject(s)
Cysteine/chemistry , I-kappa B Kinase/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Mutant Proteins/metabolism , Animals , Cells, Cultured , Cystine/chemistry , Dimerization , Humans , I-kappa B Kinase/chemistry , I-kappa B Kinase/genetics , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/genetics , Kinetics , Mice , Mice, Knockout , Mutant Proteins/chemistry , Mutant Proteins/genetics , Oxidation-Reduction , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Interaction Domains and Motifs , Protein Stability , Protein Structure, Quaternary , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Solubility , Zinc Fingers
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