Glyoxylate and pyruvate are antagonistic effectors of the Escherichia coli IclR transcriptional regulator
Glyoxylate and pyruvate are antagonistic effectors of the Escherichia coli IclR transcriptional regulator
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DOI：
10.1074/jbc.m610838200
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发表时间：
2007-06-01
影响因子：
4.8
通讯作者：
Savchenko, Alexei
中科院分区：
文献类型：
--
作者：
Lorca, Graciela L.;Ezersky, Alexandra;Savchenko, Alexei
大肠杆菌异柠檬酸裂解酶调节剂（IclR）调节乙醛酸旁路操纵子（aceBAK）的表达。IclR是一个大家庭的创始成员，它包括一个与操作符序列相互作用的DNA结合域和一个结合迄今未知小分子的c -末端结构域（C-IclR）。我们使用高通量蛋白质稳定性试验筛选了超过150个代谢支架的化学文库，以确定结合IclR的分子，然后在体外操作符结合试验中测试活性化合物。该方法鉴定的乙醛酸酯和丙酮酸酯结合C-IclR结构域的KD值分别为0.9 +/- 0.2和156.2 +/- 7.9 μ M，由等温滴定量热法确定。这两种化合物都改变了IclR与操作DNA的相互作用，但显示出拮抗作用。在体外实验中，乙醛酸酯通过促进蛋白质的非活性二聚体状态来破坏IclR/操作子复合物的形成，而丙酮酸通过稳定蛋白质的活性四聚体形式来增加IclR与aceBAK启动子的结合。在2.3 A时确定了C-IclR结构域的单独结构和与每种效应物的复合物结构，证实了这两种分子在效应物识别位点的结合，这些位点先前被表征为该家族的另一个代表，即大肠杆菌AllR调节因子。定点诱变证明了由Met- 146、Leu-154、Leu-220和Leu-143组成的疏水斑块在与效应分子相互作用中的重要性。总的来说，我们将化学筛选与功能分析和结构研究相结合的策略发现了两个具有拮抗作用的小分子，它们调节aceBAK操纵子的iclr依赖性转录。
The Escherichia coli isocitrate lyase regulator (IclR) regulates the expression of the glyoxylate bypass operon ( aceBAK). Founding member of a large family of common fold transcriptional regulators, IclR comprises a DNA binding domain that interacts with the operator sequence and a C-terminal domain ( C-IclR) that binds a hitherto unknown small molecule. We screened a chemical library of more than 150 metabolic scaffolds using a high-throughput protein stability assay to identify molecules that bind IclR and then tested the active compounds in in vitro assays of operator binding. Glyoxylate and pyruvate, identified by this method, bound the C-IclR domain with KD values of 0.9 +/- 0.2 and 156.2 +/- 7.9 mu M, as defined by isothermal titration calorimetry. Both compounds altered IclR interactions with operator DNA in electrophoretic mobility shift assays but showed an antagonistic effect. Glyoxylate disrupted the formation of the IclR/operator complex in vitro by favoring the inactive dimeric state of the protein, whereas pyruvate increased the binding of IclR to the aceBAK promoter by stabilizing the active tetrameric form of the protein. Structures of the C-IclR domain alone and in complex with each effector were determined at 2.3 A, confirming the binding of both molecules in the effector recognition site previously characterized for the other representative of the family, the E. coli AllR regulator. Site-directed mutagenesis demonstrated the importance of hydrophobic patch formed by Met- 146, Leu-154, Leu-220, and Leu-143 in interactions with effector molecules. In general, our strategy of combining chemical screens with functional assays and structural studies has uncovered two small molecules with antagonistic effects that regulate the IclR-dependent transcription of the aceBAK operon.