Protein engineering of the N-terminus of NEMO: structure stabilization and rescue of IKKβ binding.
Protein engineering of the N-terminus of NEMO: structure stabilization and rescue of IKKβ binding.
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DOI：
10.1021/bi500861x
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发表时间：
2014-11-04
期刊：
影响因子：
2.9
通讯作者：
Pellegrini, Maria
中科院分区：
文献类型：
--
作者：
Guo, Bingqian;Audu, Christopher O.;Cochran, Jared C.;Mierke, Dale F.;Pellegrini, Maria
NEMO是一种支架蛋白，与催化亚基IKKα和IKKβ一起，在IKK复合物的形成和典型NF-κB通路的激活中发挥重要作用。针对NEMO上ikk结合位点的合理药物设计将受益于结构洞察力，但迄今为止，未配体NEMO结构的确定一直受到蛋白质大小和构象异质性的阻碍。在这里，我们展示了如何利用同二聚体线圈-线圈适配器序列稳定最小IKK结合域NEMO(44-111)，并进一步了解IKK结合的结构要求。在NEMO（44-111）的n端、c端或两端加入卷曲线圈的工程构建物具有高的热稳定性和协同熔化，最重要的是，恢复了IKKβ的结合亲和力。我们通过圆二分法和核磁共振（NMR）检查了结构含量和稳定性的影响，并在荧光各向异性结合试验中测量了每个结构对IKKβ(701-745)的结合亲和力，使我们能够将结构特征和稳定性与结合亲和力联系起来。我们的研究结果提供了一种工程短稳定NEMO结构的方法，适用于核磁共振或x射线晶体学的结构表征。同时，结合亲和力的恢复表明NEMO的预定IKK结合区域与IKK结合相容，NEMO中观察到的构象异质性可能是截断的产物（44-111）。
NEMO is a scaffolding protein that, together with the catalytic subunits IKKα and IKKβ, plays an essential role in the formation of the IKK complex and in the activation of the canonical NF-κB pathway. Rational drug design targeting the IKK-binding site on NEMO would benefit from structural insight, but to date, the determination of the structure of unliganded NEMO has been hindered by protein size and conformational heterogeneity. Here we show how the utilization of a homodimeric coiled-coil adaptor sequence stabilizes the minimal IKK-binding domain NEMO(44–111) and furthers our understanding of the structural requirements for IKK binding. The engineered constructs incorporating the coiled coil at the N-terminus, C-terminus, or both ends of NEMO(44–111) present high thermal stability and cooperative melting and, most importantly, restore IKKβ binding affinity. We examined the consequences of structural content and stability by circular dichoism and nuclear magnetic resonance (NMR) and measured the binding affinity of each construct for IKKβ(701–745) in a fluorescence anisotropy binding assay, allowing us to correlate structural characteristics and stability to binding affinity. Our results provide a method for engineering short stable NEMO constructs to be suitable for structural characterization by NMR or X-ray crystallography. Meanwhile, the rescuing of the binding affinity implies that a preordered IKK-binding region of NEMO is compatible with IKK binding, and the conformational heterogeneity observed in NEMO(44–111) may be an artifact of the truncation.
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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：
8
作者：
GREENFIELD, NJ;HITCHCOCKDEGREGORI, SE
通讯作者：
HITCHCOCKDEGREGORI, SE
影响因子：
7.4
作者：
Delfín DA;Xu Y;Peterson JM;Guttridge DC;Rafael-Fortney JA;Janssen PM
通讯作者：
Janssen PM
影响因子：
15
作者：
Barth, Patrick;Schoeffler, Allyn;Alber, Tom
通讯作者：
Alber, Tom
影响因子：
11
作者：
Jin, DY;Jeang, KT
通讯作者：
Jeang, KT