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dc.contributor.authorZhou, Lien_US
dc.contributor.authorYeo, Alan T.en_US
dc.contributor.authorBallarano, Carmineen_US
dc.contributor.authorWeber, Ursen_US
dc.contributor.authorAllen, Karen N.en_US
dc.contributor.authorGilmore, Thomas D.en_US
dc.contributor.authorWhitty, Adrianen_US
dc.coverage.spatialUnited Statesen_US
dc.date.accessioned2018-02-21T15:55:18Z
dc.date.available2018-02-21T15:55:18Z
dc.date.issued2014-12-23
dc.identifierhttps://www.ncbi.nlm.nih.gov/pubmed/25400026
dc.identifier.citationLi 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.
dc.identifier.issn1520-4995
dc.identifier.urihttps://hdl.handle.net/2144/27121
dc.description.abstractHuman 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.en_US
dc.description.sponsorshipR01 GM094551 - NIGMS NIH HHS; GM094551 - NIGMS NIH HHSen_US
dc.format.extent7929 - 7944en_US
dc.languageeng
dc.relation.ispartofBiochemistry
dc.rightsCopyright © 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.en_US
dc.subjectScience & technologyen_US
dc.subjectLife sciences & biomedicineen_US
dc.subjectBiochemistry & molecular biologyen_US
dc.subjectBiologyen_US
dc.subjectCell biologyen_US
dc.subjectAnimalsen_US
dc.subjectBinding sitesen_US
dc.subjectCell lineen_US
dc.subjectDisulfidesen_US
dc.subjectHumansen_US
dc.subjectHydrogen peroxideen_US
dc.subjectI-kappa B kinaseen_US
dc.subjectIntracellular signaling peptides and proteinsen_US
dc.subjectMiceen_US
dc.subjectOxidantsen_US
dc.subjectProtein stabilityen_US
dc.subjectProtein structure, secondaryen_US
dc.subjectTNF receptor-associated factor 6en_US
dc.subjectBiochemistry and cell biologyen_US
dc.subjectMedical biochemistry and metabolomicsen_US
dc.subjectMedicinal and biomolecular chemistryen_US
dc.titleDisulfide-mediated stabilization of the IκB kinase binding domain of NF-κB essential modulator (NEMO)en_US
dc.typeArticleen_US
dc.identifier.doi10.1021/bi500920n
pubs.elements-sourcepubmeden_US
pubs.notesEmbargo: Not knownen_US
pubs.organisational-groupBoston Universityen_US
pubs.organisational-groupBoston University, College of Arts & Sciencesen_US
pubs.organisational-groupBoston University, College of Arts & Sciences, Department of Biologyen_US
pubs.organisational-groupBoston University, College of Arts & Sciences, Department of Chemistryen_US
pubs.publication-statusPublisheden_US
dc.identifier.orcid0000-0001-7296-0551 (Allen, Karen N)


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