Disulfide-mediated stabilization of the I?B kinase binding domain of NF-?B essential modulator (NEMO).
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ABSTRACT: Human NEMO (NF-?B essential modulator) is a 419 residue scaffolding protein that, together with catalytic subunits IKK? and IKK?, forms the I?B kinase (IKK) complex, a key regulator of NF-?B pathway signaling. NEMO is an elongated homodimer comprising mostly ?-helix. It has been shown that a NEMO fragment spanning residues 44-111, which contains the IKK?/? binding site, is structurally disordered in the absence of bound IKK?. 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 ?-helical coiled-coil structure that is preorganized to bind IKK? 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 IKK? in A293T cells, reconstituted TNF-induced NF-?B signaling in NEMO-deficient cells, and interacted with TRAF6. Our results indicate that the IKK? 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 IKK? binding domain in the absence of bound IKK?, thereby facilitating the structural characterization of small-molecule inhibitors.
SUBMITTER: Zhou L
PROVIDER: S-EPMC4278678 | biostudies-literature | 2014 Dec
REPOSITORIES: biostudies-literature
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