Disulfide‐Mediated Stabilization of the IκB Kinase Binding Domain of NF‐kB Essential Modulator (NEMO)

Abstract

Human NEMO is a 419 residue protein that, in complex with the catalytic subunits IKKα and IKKβ, forms the IκB kinase (IKK) complex, a key regulator of NF‐κB signaling. NEMO is a rod‐like homodimer comprising mostly α helix. It has been shown that a NEMO fragment spanning residues 44‐111, which contains the IKKα/β binding site, is structurally disordered in the absence of bound IKKβ. Here we show that enforcing dimerization of NEMO1‐120 or NEMO44‐111 constructs through introduction of one or two inter‐chain disulfide bonds – through oxidation of the endogenous Cys54 residue and/or at position 107 through a Leu107Cys mutation – induces a stable α‐helical coiled‐coil structure that is pre‐organized to bind IKKβ with high affinity. Chemical and thermal denaturation studies showed that, in the context of a covalent dimer, the ordered structure was stabilized relative to denatured state by up to 3 kcal/mol. A full‐length NEMO‐L107C protein formed covalent dimers upon treatment of mammalian cells with H2O2. Furthermore, NEMO‐L107C bound endogenous IKKβ in cells and reconstituted NF‐κB signaling in NEMO‐deficient cells. Our results indicate that IKKβ binding domain of NEMO possesses an ordered structure in unbound state, provided that it is constrained within a dimer as is the case in the constitutively dimeric full‐length NEMO protein. The stability of the NEMO coiled coil is maintained by strong inter‐helix interactions in the region centered on residue 54. The disulfide‐linked constructs we describe herein may be useful for crystallization of NEMO's IKKβ binding domain in the absence of bound IKKβ, thereby facilitating the structural characterization of small‐molecule inhibitors.