Highly Cooperative Recruitment of Ets-1 and Release of Autoinhibition by Pax5
Highly Cooperative Recruitment of Ets-1 and Release of Autoinhibition by Pax5
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DOI：
10.1016/j.jmb.2009.07.028
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发表时间：
2009-09-18
影响因子：
5.6
通讯作者：
Hagman, James
中科院分区：
文献类型：
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作者：
Fitzsimmons, Daniel;Lukin, Kara;Hagman, James
Pax5（配对盒结合因子5）是B淋巴细胞转录和谱系承诺的关键调节因子。在B细胞中，mb-1 （igg - α /免疫球蛋白相关α）启动子转录被Pax5激活，通过将e74样转化序列（Ets）家族蛋白募集到一个复合位点P5-EBS （Pax5-Ets结合位点）。此前，x射线晶体学分析揭示了Pax5和Ets-1结合P5-EBS时dna结合域之间的接触网络。在这里，我们报道Pax5通过高度合作的相互作用来组装这些三元配合物，从而克服了Ets-1的自抑制作用。利用重组蛋白，我们计算了Pax5、Ets-1和ga结合蛋白单独或一起与P5-EBS结合的K-d（app）值。Pax5本身以高亲和力（K-d等于2 nM）结合P5-EBS。Ets-1（331-440）以低亲和力（K-d = 136 nM）结合P5-EBS。然而，单独的自抑制Ets-1（280-440）不能与P5-EBS的次优序列结合。招募Ets-1（331-440）或Ets-1（280-440）与Pax5进行高效的三元配合物组装。Pax5抵消了自身抑制作用，使mb-1启动子的Ets-1结合增加了1000倍。Pax5 Gln22向丙氨酸（Q22A）的突变增强了Pax5与启动子的结合然而，Q22A极大地减少了Pax5对Ets-1（331-440）和Ets-1（280-440）的招募（分别为8.9- 300倍或bb0 - 300倍）。因此，Pax5的Gln22对于克服Ets-1自身抑制至关重要。Pax5野生型和Q22A均以相似的亲和力将ga结合蛋白α / β 1募集到mb-1启动子上，但募集效率低于Ets-1（降低了8倍左右）。我们的研究结果提示了一种允许Pax5克服Ets-1 DNA结合的自抑制的机制。总之，这些数据说明了Ets蛋白和Pax5之间的伙伴关系。2009爱思唯尔有限公司版权所有。
Pax5 (paired box binding factor 5) is a critical regulator of transcription and lineage commitment in B lymphocytes. In B cells, mb-1 (Ig-alpha/immunoglobulin-associated alpha) promoter transcription is activated by Pax5 through its recruitment of E74-like transforming sequence (Ets) family proteins to a composite site, the P5-EBS (Pax5-Ets binding site). Previously, X-ray crystallographic analysis revealed a network of contacts between the DNA-binding domains of Pax5 and Ets-1 while bound to the P5-EBS. Here, we report that Pax5 assembles these ternary complexes via highly cooperative interactions that overcome the autoinhibition of Ets-1. Using recombinant proteins, we calculated K-d(app) values for the binding of Pax5, Ets-1, and GA-binding proteins, separately or together, to the P5-EBS. By itself, Pax5 binds the P5-EBS with high affinity (K-d congruent to 2 nM). Ets-1 (331-440) bound the P5-EBS by itself with low affinity (K-d = 136 nM). However, autoinhibited Ets-1(280-440) alone does not bind detectably to the suboptimal sequences of the P5-EBS. Recruitment of Ets-1(331-440) or Ets-1(280-440) resulted in highly efficient ternary complex assembly with Pax5. Pax5 counteracts autoinhibition and increases binding of Ets-1 of the mb-1 promoter by > 1000-fold. Mutation of Pax5 Gln22 to alanine (Q22A) enhances promoter binding by Pax5; however, Q22A greatly reduces recruitment of Ets-1(331-440) and Ets-1(280-440) by Pax5 (8.9- or >300-fold, respectively). Thus, Gln22 of Pax5 is essential for overcoming Ets-1 autoinhibition. Pax5 wild type and Q22A each recruited GA-binding protein alpha/beta 1 to the mb-1 promoter with similar affinities, but recruitment was less efficient than that of Ets-1 (reduced by similar to 8-fold). Our results suggest a mechanism that allows Pax5 to overcome autoinhibition of Ets-1 DNA binding. In summary, these data illustrate requirements for partnerships between Ets proteins and Pax5. (C) 2009 Elsevier Ltd. All rights reserved.