MECHANISM OF AUTOPHOSPHORYLATION OF ESCHERICHIA-COLI NITROGEN REGULATOR-II (NR(II) OR NTR(B)) - TRANS-PHOSPHORYLATION BETWEEN SUBUNITS

Nitrogen regulator II (NR(II) or NtrB) is a homodimeric signal-transdu cing protein kinase/phosphatase responsible for the transcriptional re gulation 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) autop hosphorylation by using purified components. Alteration of the site of NR(II) autophosphorylation to asparagine (H-139-->N [H139N]) or delet ion of the C-terminal 59 amino acids of NR(II) (ter291) resulted in pr oteins that were not autophosphorylated upon incubation with ATP. Alte ration of glycine 313 to alanine resulted in a protein (G313A) that wa s phosphorylated to a lesser extent than the wild-type protein. Unlike wild-type NR(II) and H139N, G313A could not be efficiently cross-link ed to [alpha-P-32]ATP, suggesting that the G313A mutation affects nucl eotide binding. Fusion of maltose-binding protein (MBP) to the N-termi nal end or NR(II) resulted in a protein (MBP-NR(II)) that autophosphor ylated normally. We developed a procedure for forming mixed dimers in vitro from these proteins. In mixed dimers consisting of MBP-NR(II) an d H139N, only the MBP-NR(II) subunit is phosphorylated. In contrast, i n mixed dimers consisting of MBP-NR(II) and G313A, phosphorylation is predominantly on the G313A subunit. We also demonstrated that the G313 A and H139N proteins could complement for the autophosphorylation reac tion when they were treated so as to permit the formation of mixed dim ers and that the wild-type and H139N proteins could phosphorylate the ter291 protein. These results indicate that the autophosphorylation re action occurs within the dimer by a trans, intersubunit mechanism in w hich one subunit binds ATP and phosphorylates the other subunit.