NEMO is an essential regulatory component of the IκB kinase (IKK) complex, which controls activation of the NF-κB signaling pathway. Herein, we show that NEMO exists as a disulfide-bonded dimer when isolated from several cell types and analyzed by SDS-polyacrylamide gel electrophoresis under non-reducing conditions. Treatment of cells with hydrogen peroxide (H 2 O 2 ) induces further formation of NEMO dimers. Disulfide bond-mediated formation of NEMO dimers requires Cys54 and Cys347. The ability of these residues to form disulfide bonds is consistent with their location in a NEMO dimer structure that we generated by molecular modeling. We also show that pretreatment with H 2 O 2 decreases TNFα-induced IKK activity in NEMO-reconstituted cells, and that TNFα has a diminished ability to activate NF-κB DNA binding in cells reconstituted with NEMO mutant C54/347A. This study implicates NEMO as a target of redox regulation and presents the first structural model for the NEMO protein.

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 NEMO and its thermal stability were independent of the presence or absence of inter-chain disulfide bonds.

NF‐κB essential modulator (NEMO) is a major component of the Iκ Kinase (IKK) complex, which serves as a critical component of NF‐κB signaling. In the IKK complex, NEMO forms a 2:2 interaction with its binding partner Iκ Kinase β (IKKβ). Small molecule inhibitors that specifically block the interaction of NEMO with IKKβ would have potential utility in inflammatory diseases and certain types of cancer. Generating a recombinant form of full‐length NEMO for biochemical characterization and inhibitor discovery has proven to be problematic, however, due to disulfide‐mediated misfolding and aggregation involving some of NEMO's 11 cysteine residues. We show here that highly soluble and homogeneous full‐length NEMO can be generated by mutating five or seven non‐critical cysteine residues. Both the 5xAla and 7xAla mutants show wild‐type affinity for IKKβ in a fluorescence anisotropy binding assay. Morevover, when transfected into NEMO −/− fibroblasts, both mutants were able to restore NF‐κB‐dependent cell survival in response to TNFα treatment. Our results show that none of the seven mutated cysteines are required for the IKKβ binding activity of NEMO, and identify a form of full‐length recombinant protein suitable for screening assays and structural characterization. (This work was supported by NIH GM094551 to AW and TDG)