Disulfide-mediated stabilization of the IκB kinase binding domain of NF-κB essential modulator (NEMO).
Disulfide-mediated stabilization of the IκB kinase binding domain of NF-κB essential modulator (NEMO).
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DOI：
10.1021/bi500920n
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发表时间：
2014-12-23
期刊：
影响因子：
2.9
通讯作者：
Whitty, Adrian
中科院分区：
文献类型：
--
作者：
Zhou, Li;Yeo, Alan T.;Ballarano, Carmine;Weber, Urs;Allen, Karen N.;Gilmore, Thomas D.;Whitty, Adrian
人NEMO（NF-κB必需调节因子）是一种419个残基的支架蛋白，与催化亚基IKKα和IKKβ一起形成IκB激酶（IKK）复合物，该复合物是NF-κB通路信号传导的关键调节因子。NEMO是主要包含α-螺旋的伸长的同源二聚体。研究表明，包含IKKα/β结合位点的跨越残基44-111的NEMO片段在不存在结合IKKβ的情况下结构紊乱。在此，我们发现，通过引入一个或两个链间二硫键，通过天然Cys 54残基的氧化和/或通过Leu 107 Cys突变在107位处，强制NEMO 1 -120或NEMO 44 -111构建体的二聚化，诱导稳定的α-螺旋卷曲螺旋结构，该结构被预先组织以高亲和力结合IKKβ。化学和热变性研究表明，在共价二聚体的情况下，有序结构相对于变性状态稳定高达3千卡/摩尔。一个全长NEMO-L107 C蛋白质形成共价二聚体后，用过氧化氢处理的哺乳动物细胞。此外，NEMO-L107 C在A293 T细胞中结合内源性IKKβ，在NEMO缺陷细胞中重建TNF诱导的NF-κB信号传导，并与TRAF 6相互作用。我们的研究结果表明，IKKβ结合结构域的NEMO在未结合状态下具有有序的结构，只要它被限制在一个二聚体的情况下，在组成性二聚体全长NEMO蛋白。NEMO卷曲螺旋的稳定性通过以残基54为中心的区域中的强螺旋间相互作用来维持。我们在此描述的二硫键连接的构建体可用于在不存在结合的IKKβ的情况下结晶NEMO的IKKβ结合结构域，从而促进小分子抑制剂的结构表征。
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.
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影响因子：
15
作者：
Golden, Mary S.;Cote, Shaun M.;Sayeg, Marianna;Zerbe, Brandon S.;Villar, Elizabeth A.;Beglov, Dmitri;Sazinsky, Stephen L.;Georgiadis, Rosina M.;Vajda, Sandor;Kozakov, Dima;Whitty, Adrian
通讯作者：
Whitty, Adrian
DOI：
10.1074/jbc.m109.099895
发表时间：
2010-04-30
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Baima ET;Guzova JA;Mathialagan S;Nagiec EE;Hardy MM;Song LR;Bonar SL;Weinberg RA;Selness SR;Woodard SS;Chrencik J;Hood WF;Schindler JF;Kishore N;Mbalaviele G
通讯作者：
Mbalaviele G
影响因子：
3.5
作者：
Gautheron, Jeremie;Pescatore, Alessandra;Courtois, Gilles
通讯作者：
Courtois, Gilles
DOI：
10.1073/pnas.0800452105
发表时间：
2008-05-27
影响因子：
11.1
作者：
Chang, Chia-en A.;McLaughlin, William A.;McCammon, J. Andrew
通讯作者：
McCammon, J. Andrew
影响因子：
14.8
作者：
Greenfield, Norma J.
通讯作者：
Greenfield, Norma J.