MECHANISM OF AUTOPHOSPHORYLATION OF ESCHERICHIA-COLI NITROGEN REGULATOR-II (NR(II) OR NTR(B)) - TRANS-PHOSPHORYLATION BETWEEN SUBUNITS
MECHANISM OF AUTOPHOSPHORYLATION OF ESCHERICHIA-COLI NITROGEN REGULATOR-II (NR(II) OR NTR(B)) - TRANS-PHOSPHORYLATION BETWEEN SUBUNITS
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DOI：
10.1128/jb.175.21.7024-7032.1993
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发表时间：
1993-11-01
影响因子：
3.2
通讯作者：
NINFA, AJ
中科院分区：
文献类型：
--
作者：
NINFA, EG;ATKINSON, MR;NINFA, AJ
氮调节子 II（NR(II) 或 NtrB）是一种同二聚体信号转导蛋白激酶/磷酸酶，负责大肠杆菌中 Ntr 调节子的转录调节。 NR(II) 是蛋白质大家族的成员，该蛋白质大家族是相关双组分信号转导系统的一部分。我们利用纯化的组分研究了 NR(II) 自磷酸化的机制。将 NR(II) 自磷酸化位点改变为天冬酰胺 (H-139-->N [H139N]) 或删除 NR(II) (ter291) C 端 59 个氨基酸会导致蛋白质在与 ATP 孵育时不会自磷酸化。将甘氨酸 313 改变为丙氨酸导致蛋白质 (G313A) 的磷酸化程度低于野生型蛋白质。与野生型 NR(II) 和 H139N 不同，G313A 无法与 [α-P-32]ATP 有效交联，表明 G313A 突变影响核苷酸结合。麦芽糖结合蛋白 (MBP) 与 N 末端或 NR(II) 融合，产生正常自磷酸化的蛋白质 (MBP-NR(II))。我们开发了一种在体外从这些蛋白质形成混合二聚体的方法。 在由 MBP-NR(II) 和 H139N 组成的混合二聚体中，仅 MBP-NR(II) 亚基被磷酸化。相反，在由 MBP-NR(II) 和 G313A 组成的混合二聚体中，磷酸化主要发生在 G313A 亚基上。我们还证明，当 G313A 和 H139N 蛋白经过处理以允许形成混合二聚体时，它们可以补充自磷酸化反应，并且野生型和 H139N 蛋白可以磷酸化 ter291 蛋白。这些结果表明，自磷酸化反应通过反式亚基间机制在二聚体内发生，其中一个亚基结合 ATP 并使另一个亚基磷酸化。
Nitrogen regulator II (NR(II) or NtrB) is a homodimeric signal-transducing protein kinase/phosphatase responsible for the transcriptional regulation of the Ntr regulon in Escherichia coli. NR(II) is a member of a large family of proteins that are part of the related two-component signal transduction systems. We studied the mechanism of NR(II) autophosphorylation by using purified components. Alteration of the site of NR(II) autophosphorylation to asparagine (H-139-->N [H139N]) or deletion of the C-terminal 59 amino acids of NR(II) (ter291) resulted in proteins that were not autophosphorylated upon incubation with ATP. Alteration of glycine 313 to alanine resulted in a protein (G313A) that was phosphorylated to a lesser extent than the wild-type protein. Unlike wild-type NR(II) and H139N, G313A could not be efficiently cross-linked to [alpha-P-32]ATP, suggesting that the G313A mutation affects nucleotide binding. Fusion of maltose-binding protein (MBP) to the N-terminal end or NR(II) resulted in a protein (MBP-NR(II)) that autophosphorylated normally. We developed a procedure for forming mixed dimers in vitro from these proteins. In mixed dimers consisting of MBP-NR(II) and H139N, only the MBP-NR(II) subunit is phosphorylated. In contrast, in mixed dimers consisting of MBP-NR(II) and G313A, phosphorylation is predominantly on the G313A subunit. We also demonstrated that the G313A and H139N proteins could complement for the autophosphorylation reaction when they were treated so as to permit the formation of mixed dimers and that the wild-type and H139N proteins could phosphorylate the ter291 protein. These results indicate that the autophosphorylation reaction occurs within the dimer by a trans, intersubunit mechanism in which one subunit binds ATP and phosphorylates the other subunit.