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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.

Mutation of nonessential cysteines shows that the NF-κB essential modulator forms a constitutive noncovalent dimer that binds IκB kinase-β with high affinity.

Biochemistry (2013-11-26)
Shaun M Cote, Thomas D Gilmore, Robert Shaffer, Urs Weber, Rishitha Bollam, Mary S Golden, Kimberley Glover, Melanie Herscovitch, Thomas Ennis, Karen N Allen, Adrian Whitty
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

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