NEMO (NF-κB essential modulator) associates with 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 about 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 a K(D) of 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 five-Ala and seven-Ala NEMO mutants can 
interact with cellular IKKβ 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 IKKβ binding affinity of NEMO is unaffected by the presence or 
absence of interchain 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 NEMO and its thermal stability were independent of the presence or 
absence of interchain disulfide bonds.