title: The amino-terminal GAF domain of Azotobacter vinelandii NifA binds 2-oxoglutarate to resist inhibition by NifL under nitrogen-limiting conditions.
abstract: The expression of genes required for the synthesis of molybdenum nitrogenase in Azotobacter vinelandii is controlled by the NifL-NifA transcriptional regulatory complex in response to nitrogen, carbon, and redox status. Activation of nif gene expression by the transcriptional activator NifA is inhibited by direct protein-protein interaction with NifL under conditions unfavorable for nitrogen fixation. We have recently shown that the NifL-NifA system responds directly to physiological concentrations of 2-oxoglutarate, resulting in relief of NifA activity from inhibition by NifL under conditions when fixed nitrogen is limiting. The inhibitory activity of NifL is restored under conditions of excess combined nitrogen through the binding of the signal transduction protein Av GlnK to the carboxyl-terminal domain of NifL. The amino-terminal domain of NifA comprises a GAF domain implicated in the regulatory response to NifL. A truncated form of NifA lacking this domain is not responsive to 2-oxoglutarate in the presence of NifL, suggesting that the GAF domain is required for the response to this ligand. Using isothermal titration calorimetry, we demonstrate stoichiometric binding of 2-oxoglutarate to both the isolated GAF domain and the full-length A. vinelandii NifA protein with a dissociation constant of approximately 60 microm. Limited proteolysis experiments indicate that the binding of 2-oxoglutarate increases the susceptibility of the GAF domain to trypsin digestion and also prevents NifL from protecting these cleavage sites. However, protection by NifL is restored when the non-modified (non-uridylylated) form of Av GlnK is also present. Our results suggest that the binding of 2-oxoglutarate to the GAF domain of NifA may induce a conformational change that prevents inhibition by NifL under conditions when fixed nitrogen is limiting.
authors.affiliation: Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, United Kingdom.
authors.forename: Richard
authors.initials: R
authors.lastname: Little
authors.forename: Ray
authors.initials: R
authors.lastname: Dixon
mesh_terms: D016948:Azotobacter vinelandii / Q000737:chemistry*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D001665:Binding Sites; D007656:Ketoglutaric Acids / Q000378:metabolism*; D009584:Nitrogen / Q000008:administration & dosage*; D009586:Nitrogen Fixation; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation / Q000187:drug effects; D015398:Signal Transduction; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D007656:Ketoglutaric Acids; C061338:NifA protein, Bacteria; D010446:Peptide Fragments; D014157:Transcription Factors; C084296:nifL protein, Bacteria; D014357:Trypsin; D009584:Nitrogen