title: Mechanism of autophosphorylation of Escherichia coli nitrogen regulator II (NRII or NtrB): trans-phosphorylation between subunits.
abstract: 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.
authors.affiliation: Department of Biochemistry, Wayne State University School of Medicine, Detroit, Michigan 48201.
authors.forename: E G
authors.initials: EG
authors.lastname: Ninfa
authors.forename: M R
authors.initials: MR
authors.lastname: Atkinson
authors.forename: E S
authors.initials: ES
authors.lastname: Kamberov
authors.forename: A J
authors.initials: AJ
authors.lastname: Ninfa
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial; D005809:Genes, Regulator; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051922:PII Nitrogen Regulatory Proteins; D010766:Phosphorylation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D046911:Macromolecular Substances; D051922:PII Nitrogen Regulatory Proteins; D011994:Recombinant Proteins; C404143:PIID regulatory protein, Bacteria