Disulfide-Mediated Stabilization of the IκB Kinase Binding Domain of NF-κB Essential Modulator (NEMO)

Abstract

Human NEMO (NF-κB essential modulator) is a 419 residue scaffolding protein that, together with catalytic subunits IKKα and IKKβ, forms the IκB kinase (IKK) complex, a key regulator of NF-κB pathway signaling. NEMO is an elongated 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. Herein we show that enforcing dimerization of NEMO1–120 or NEMO44–111 constructs through introduction of one or two interchain disulfide bonds, through oxidation of the native Cys54 residue and/or at position 107 through a Leu107Cys mutation, induces a stable α-helical coiled-coil structure that is preorganized 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 the 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 A293T cells, reconstituted TNF-induced NF-κB signaling in NEMO-deficient cells, and interacted with TRAF6. Our results indicate that the IKKβ binding domain of NEMO possesses an ordered structure in the 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 interhelix 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.

Mitochondrial reactive oxygen species enable proinflammatory signaling through disulfide linkage of NEMO

“…In particular, the precise composition of the covalently linked 200-kDa complex that is formed upon infection through the formation of the disulfide bonds between NEMO molecules remains to be investigated. Cys 54 and Cys 347 have been shown to mediate disulfide linkage of NEMO monomers into a covalently linked dimer of about 100 kDa in H 2 O 2 -treated cells (33)(34)(35). Our data now show that, in infected cells, Cys 54 and Cys 347 mediate additional covalent interactions.…”

“…The primary functional unit of NEMO is a constitutive, noncovalently linked dimer. Covalent links between NEMO monomers formed by disulfide bonds between the redox-sensitive cysteines Cys 54 and Cys 347 have been shown to stabilize the NEMO dimer and increase its affinity to IKK in vitro (33)(34)(35). Therefore, we investigated whether mtROS produced by L. monocytogenes-infected macrophages enabled IKK complex activation by inducing disulfide bondmediated covalent linkage of NEMO dimers.…”

The IKK-binding domain of NEMO is an irregular coiled coil with a dynamic binding interface

“…NEMO-EEAA and NEMO-I65M show a large improvement in folding, stability and IKKβ binding affinity compared to the wild type fragment of NEMO (44–111) 22 . Notably, a similar approach of enforcing the dimerization of NEMO (44–111) through oxidation at position 107 (L107C mutation) was reported to induce a stable α-helical coiled-coil structure that is preorganized to bind IKKβ with high affinity 36 . The results can be interpreted to indicate that the IKKβ binding domain of NEMO possesses an ordered structure in the unbound state when part of full length NEMO, but becomes conformationally heterogenous and unstable when truncated below residue 130.…”

Production, Crystallization, and Structure Determination of the IKK-binding Domain of NEMO

“…Although other constructs stabilizing the ordered structure of the IKK-binding domain of NEMO through cysteine disulfide linkage and binding IKKβ with high affinity have been described 30 , the protocol here described represents the first successful approach to structure determination of the IKK-binding domain of NEMO in the unbound form.…”