The response regulator OmpR oligomerizes via β-sheets to form head-to-head dimers
The response regulator OmpR oligomerizes via β-sheets to form head-to-head dimers
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DOI：
10.1016/j.jmb.2005.05.057
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发表时间：
2005-07-29
影响因子：
5.6
通讯作者：
Kenney, LJ
中科院分区：
文献类型：
--
作者：
Maris, AE;Walthers, D;Kenney, LJ
在大肠杆菌中，EnvZ/OmpR双组分调节系统调节孔蛋白基因ompF和ompC的表达以响应渗透压的变化。最近已经变得明显的是，OmpR通过调节除了孔蛋白基因之外的许多基因的表达而作为全局调节剂起作用。OmpR由两个结构域组成; N-末端受体结构域的磷酸化增加了C-末端结构域的DNA结合亲和力，反之亦然。许多反应调节剂包括PhoB和FixJ在磷酸化后二聚化。在这里，我们表明，OmpR二聚化是由磷酸化或DNA结合刺激。这里揭示的二聚化界面是意料之外的，以前也没有预测到。使用公认的头-尾串联结合模型作为指导，我们着手研究通过蛋白质-蛋白质交联方法与DNA结合的OmpR二聚体之间的分子间相互作用。令人惊讶的是，我们预期形成交联二聚体的氨基酸位置没有。相反，预测不会形成二聚体的位置却形成了二聚体。由于这些结果，我们设计了一系列23个半胱氨酸取代的OmpR突变体，用于研究通过β折叠区形成的二聚体界面。这种四链β折叠是有翼螺旋-转角-螺旋蛋白OmpR组的独特特征。许多半胱氨酸取代的突变体对野生型OmpR是显性的，被乙酰磷酸以及同源激酶EnvZ磷酸化，并且交联蛋白能够结合DNA。我们的研究结果是一致的模型中，OmpR结合到DNA的头对头的方向，在以前提出的不对称头到尾模型。他们还提出了一种可能性，即OmpR可能能够采用一种以上的方向，因为它与大量基因结合以激活或抑制转录。(c)2005爱思唯尔有限公司保留所有权利。
In Escherichia coli, the EnvZ/OmpR two-component regulatory system regulates expression of the porin genes ompF and ompC in response to changes in osmolarity. It has recently become apparent that OmpR functions as a global regulator, by regulating the expression of many genes in addition to the porin genes. OmpR consists of two domains; phosphorylation of the N-terminal receiver domain increases DNA binding affinity of the C-terminal domain and vice versa. Many response regulators including PhoB and FixJ dimerize upon phosphorylation. Here, we demonstrate that OmpR dimerization is stimulated by phosphorylation or by DNA binding. The dimerization interface revealed here was unanticipated and had previously not been predicted. Using the accepted head-to-tail tandem-binding model as a guide, we set out to examine the intermolecular interactions between OmpR dimers bound to DNA by protein-protein cross-linking methods. Surprisingly, amino acid positions that we expected to form cross-linked dimers did not. Conversely, positions predicted not to form dimers did. Because of these results, we designed a series of 23 cysteine-substituted OmpR mutants that were used to investigate dimer interfaces formed via the beta-sheet region. This four-stranded beta-sheet is a unique feature of the OmpR group of winged helix-turn-helix proteins. Many of the cysteine-substituted mutants are dominant to wild-type OmpR, are phosphorylated by acetyl phosphate as well as the cognate kinase EnvZ, and the cross-linked proteins are capable of binding to DNA. Our results are consistent with a model in which OmpR binds to DNA in a head-to-head orientation, in contrast to the previously proposed asymmetric head-to-tail model. They also raise the possibility that OmpR may be capable of adopting more than one orientation as it binds to a vast array of genes to activate or repress transcription. (c) 2005 Elsevier Ltd. All rights reserved.