Mutation of Non‐Essential Cysteines Leads to Highly Soluble and Active Recombinant Full‐Length NEMO

Abstract

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)