Disulfide-mediated stabilization of the IκB kinase binding domain of NF-κB essential modulator (NEMO).
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.
Department of Chemistry and ‡Department of Biology, Boston University , Boston, Massachusetts 02215, United States.
Li
L
Zhou
Alan T
AT
Yeo
Carmine
C
Ballarano
Urs
U
Weber
Karen N
KN
Allen
Thomas D
TD
Gilmore
Adrian
A
Whitty
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D051550:I-kappa B Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D016877:Oxidants / Q000494:pharmacology; D055550:Protein Stability / Q000187:drug effects; D017433:Protein Structure, Secondary; D048029:TNF Receptor-Associated Factor 6 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D004220:Disulfides; C491731:IKBKG protein, human; D047908:Intracellular Signaling Peptides and Proteins; C514981:NEMO protein, mouse; D016877:Oxidants; D048029:TNF Receptor-Associated Factor 6; D006861:Hydrogen Peroxide; C496559:CHUK protein, human; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496562:Ikbkb protein, mouse