title: Mechanism of activation of a response regulator: interaction of NtrC-P dimers induces ATPase activity.
abstract: NtrC is the transcriptional activator for nitrogen-regulated promoters and, as a response regulator, belongs to the protein family of two-component systems. The activity of all response regulators is modulated by phosphorylation of the conserved N-terminal receiver domain. Phosphorylation of the dimeric NtrC has two consequences: (i) a strong increase in the cooperative binding of NtrC to two adjacent binding sites and (ii) activation of NtrC as an ATPase. Here we show that phosphorylation of NtrC is not sufficient for activation of NtrC. At low protein concentrations (50 nM), phosphorylated NtrC was only active as an ATPase upon cooperative binding to DNA. At high protein concentrations (above 50 nM), NtrC was active in the absence of DNA, and activation occurred in parallel with the formation of high-molecular-weight aggregates. We infer that activation of NtrC involves an interaction between two NtrC-P dimers and proceeds in two steps. The first step is the phosphorylation of NtrC. The second step is the interaction between two NtrC-P dimers. This interaction induces the conformational change in NtrC-P to the active conformation.
authors.affiliation: Department of Biology, University of Konstanz, Germany.
authors.forename: I
authors.initials: I
authors.lastname: Mettke
authors.forename: U
authors.initials: U
authors.lastname: Fiedler
authors.forename: V
authors.initials: V
authors.lastname: Weiss
mesh_terms: D000251:Adenosine Triphosphatases / Q000187:drug effects / Q000378:metabolism*; D001360:Avidin / Q000494:pharmacology; D001426:Bacterial Proteins / Q000187:drug effects / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins / Q000187:drug effects / Q000378:metabolism*; D004789:Enzyme Activation; D004795:Enzyme Stability; D008958:Models, Molecular; D008969:Molecular Sequence Data; D051922:PII Nitrogen Regulatory Proteins; D010750:Phosphoproteins / Q000187:drug effects / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D015534:Trans-Activators; D014157:Transcription Factors
chemical_list: D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D051922:PII Nitrogen Regulatory Proteins; D010750:Phosphoproteins; D015534:Trans-Activators; D014157:Transcription Factors; D001360:Avidin; D010744:Phosphoric Monoester Hydrolases; D000251:Adenosine Triphosphatases