Interaction of the IkappaBalpha C-terminal PEST sequence with NF-kappaB: insights into the inhibition of NF-kappaB DNA binding by IkappaBalpha.
Interaction of the IkappaBalpha C-terminal PEST sequence with NF-kappaB: insights into the inhibition of NF-kappaB DNA binding by IkappaBalpha.
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DOI：
10.1016/j.jmb.2009.03.048
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发表时间：
2009-05-15
影响因子：
5.6
通讯作者：
Dyson, H. Jane
中科院分区：
文献类型：
--
作者：
Sue, Shih-Che;Dyson, H. Jane
转录因子NF-κB （p50/p65）可以结合κB DNA元件或其抑制蛋白i -κB α，但这两种结合事件是相互排斥的。从现有的晶体结构数据来看，这种排他性的原因并不明显。i -κB α的c端pest样序列似乎参与了这一过程，但它位于i -κB α/NF-κB复合物的两个已发表的x射线结构中，距离NF-κB DNA结合域的DNA接触环很远。我们利用核磁共振波谱和差分同位素标记技术研究了溶液中p50/p65 NF-κB异二聚体与i -κB α的相互作用。我们的测量能够解决两种晶体结构之间的局部结构差异，我们证实了IκBα PEST结构域的主要相互作用是与p65亚基的dna结合结构域。DNA接触序列中关键精氨酸残基的突变导致PEST序列与p65亚结构域的特异性相互作用丧失。我们得出结论，在PEST序列区域的i -κB α/NF-κB复合物的局部结构与p65中酸性序列与基本DNA接触序列的直接相互作用是一致的，从而通过竞争机制降低了NF-κB对DNA的亲和力，这一机制仍有待充分阐明。
The transcription factor NF-κB (p50/p65) binds either a κB DNA element or its inhibitor protein, IκBα, but these two binding events are mutually exclusive. The reason for this exclusivity is not obvious from the available crystal structure data. The C-terminal PEST-like sequence of IκBα appears to be involved in the process, but it is located in both of the published X-ray structures of the IκBα/NF-κB complex at a significant distance away from the DNA contact loop in the NF-κB DNA-binding domain. We have used nuclear magnetic resonance spectroscopy and differential isotopic labeling to probe the interactions between the p50/p65 NF-κB heterodimer and IκBα in solution. Our measurements are able to resolve a local structural discrepancy between the two crystal structures, and we confirm that the primary interaction of the IκBα PEST domain is with the DNA-binding domain of the p65 subunit. Mutagenesis of key arginine residues in the DNA contact sequence results in the loss of specific interaction of the PEST sequence with the p65 subdomain. We conclude that the local structure of the IκBα/NF-κB complex in the region of the PEST sequence is consistent with a direct interaction of this acidic sequence with the basic DNA contact sequence in p65, thus reducing the affinity of NF-κB for DNA by a competitive mechanism that is still to be elucidated fully.
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