Mechanism of autophosphorylation of Escherichia coli nitrogen regulator II (NR(II) or NtrB): trans-Phosphorylation between subunits
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 [α-32P]ATP, suggesting that the G313A mutation affects nucleotide binding. Fusion of maltose-binding protein (MBP) to the N- terminal end of 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.
Ninfa, EG; Atkinson, MR; Kamberov, ES; Ninfa, AJ
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