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    Disulfide-mediated stabilization of the IκB kinase binding domain of NF-κB essential modulator (NEMO)

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    Date Issued
    2014-12-23
    Publisher Version
    10.1021/bi500920n
    Author(s)
    Zhou, Li
    Yeo, Alan T.
    Ballarano, Carmine
    Weber, Urs
    Allen, Karen N.
    Gilmore, Thomas D.
    Whitty, Adrian
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    Permanent Link
    https://hdl.handle.net/2144/27121
    Citation (published version)
    Li Zhou, Alan T Yeo, Carmine Ballarano, Urs Weber, Karen N Allen, Thomas D Gilmore, Adrian Whitty. 2014. "Disulfide-mediated stabilization of the IκB kinase binding domain of NF-κB essential modulator (NEMO).." Biochemistry, Volume 53, Issue 50, pp. 7929 - 7944.
    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.
    Rights
    Copyright © 2014 American Chemical Society. This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.
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    • BU Open Access Articles [3664]
    • CAS: Chemistry: Scholarly Papers [120]


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