Maris Ann E, Walthers Don, Mattison Kirsten, Byers Nicole, Kenney Linda J
Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095-1569, USA.
J Mol Biol. 2005 Jul 29;350(5):843-56. doi: 10.1016/j.jmb.2005.05.057.
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.
在大肠杆菌中，EnvZ/OmpR双组分调节系统可根据渗透压的变化调节孔蛋白基因ompF和ompC的表达。最近发现，OmpR除了调节孔蛋白基因外，还通过调节许多基因的表达而发挥全局调节因子的作用。OmpR由两个结构域组成；N端接收结构域的磷酸化会增加C端结构域的DNA结合亲和力，反之亦然。包括PhoB和FixJ在内的许多应答调节因子在磷酸化后会形成二聚体。在此，我们证明OmpR二聚化受到磷酸化或DNA结合的刺激。此处揭示的二聚化界面出人意料，此前并未被预测到。以公认的头对头串联结合模型为指导，我们着手通过蛋白质-蛋白质交联方法研究与DNA结合的OmpR二聚体之间的分子间相互作用。令人惊讶的是，我们预期会形成交联二聚体的氨基酸位置并未形成。相反，预测不会形成二聚体的位置却形成了。基于这些结果，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折叠区域形成的二聚体界面。这种四链β折叠是有翼螺旋-转角-螺旋蛋白OmpR家族的独特特征。许多半胱氨酸取代的突变体对野生型OmpR具有显性作用，能被乙酰磷酸以及同源激酶EnvZ磷酸化，并且交联后的蛋白能够结合DNA。我们的结果与一个模型相符，即OmpR以头对头的方向结合DNA，这与之前提出的不对称头对尾模型相反。这些结果还提出了一种可能性，即OmpR在结合大量基因以激活或抑制转录时，可能能够采用不止一种方向。
