Ninfa E G, Atkinson M R, Kamberov E S, Ninfa A J
Department of Biochemistry, Wayne State University School of Medicine, Detroit, Michigan 48201.
J Bacteriol. 1993 Nov;175(21):7024-32. doi: 10.1128/jb.175.21.7024-7032.1993.
Nitrogen regulator II (NRII or NtrB) is a homodimeric signal-transducing protein kinase/phosphatase responsible for the transcriptional regulation of the Ntr regulon in Escherichia coli. NRII 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 NRII autophosphorylation by using purified components. Alteration of the site of NRII autophosphorylation to asparagine (H-139-->N [H139N]) or deletion of the C-terminal 59 amino acids of NRII (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 NRII and H139N, G313A could not be efficiently cross-linked to [alpha-32P]ATP, suggesting that the G313A mutation affects nucleotide binding. Fusion of maltose-binding protein (MBP) to the N-terminal end of NRII resulted in a protein (MBP-NRII) that autophosphorylated normally. We developed a procedure for forming mixed dimers in vitro from these proteins. In mixed dimers consisting of MBP-NRII and H139N, only the MBP-NRII subunit is phosphorylated. In contrast, in mixed dimers consisting of MBP-NRII 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.
氮调节蛋白II（NRII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操纵子的转录调控。NRII是一大类蛋白质家族的成员，这些蛋白质是相关双组分信号转导系统的一部分。我们使用纯化的组分研究了NRII自身磷酸化的机制。将NRII自身磷酸化位点改变为天冬酰胺（H-139→N [H139N]）或缺失NRII的C末端59个氨基酸（ter291），导致蛋白质在与ATP孵育时不能进行自身磷酸化。将甘氨酸313改变为丙氨酸导致一种蛋白质（G313A），其磷酸化程度低于野生型蛋白质。与野生型NRII和H139N不同，G313A不能有效地与[α-32P]ATP交联，这表明G313A突变影响核苷酸结合。将麦芽糖结合蛋白（MBP）融合到NRII的N末端产生一种蛋白质（MBP-NRII），其能正常地进行自身磷酸化。我们开发了一种在体外由这些蛋白质形成混合二聚体的方法。在由MBP-NRII和H139N组成的混合二聚体中，只有MBP-NRII亚基被磷酸化。相反，在由MBP-NRII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蛋白质经过处理以允许形成混合二聚体时，它们可以互补自身磷酸化反应，并且野生型和H139N蛋白质可以磷酸化ter291蛋白质。这些结果表明，自身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。
