title: Phosphorylation-induced dimerization of the FixJ receiver domain.
abstract: The 'two-component' transcriptional activator FixJ controls nitrogen fixation in Sinorhizobium meliloti. Phosphorylation of FixJ induces its dimerization, as evidenced by gel permeation chromatography and equilibrium sedimentation analysis. Phosphorylation-induced dimerization is an intrinsic property of the isolated receiver domain FixJN. Accordingly, chemical phosphorylation of both FixJ and FixJN are second-order reactions with respect to protein concentration. However, the second-order phosphorylation constant is 44-fold higher for FixJN than for FixJ. Therefore, the C-terminal transcriptional activator domain FixJC inhibits the chemical phosphorylation of the receiver domain FixJN. Conversely, FixJN has been shown previously to inhibit FixJC activity approximately 40-fold, reflecting the interaction between FixJN and FixJC. Therefore, we propose that modulation of FixJ activity involves both its dimerization and the disruption of the interface between FixJN and FixJC, resulting in the opening of the protein structure. Alanine scanning mutagenesis of FixJN indicated that the FixJ approximately P dimerization interface involves Val-91 and Lys-95 in helix alpha4. Dimerization was required for high-affinity binding to fixK promoter DNA.
authors.affiliation: Laboratoire de Biologie Moléculaire des Relations Plantes-Microorganismes, UMR 215 INRA-CNRS, Chemin de Borde Rouge, BP 27, 31326 Castanet-Tolosan Cedex, France.
authors.forename: S
authors.initials: S
authors.lastname: Da Re
authors.forename: J
authors.initials: J
authors.lastname: Schumacher
authors.forename: P
authors.initials: P
authors.lastname: Rousseau
authors.forename: J
authors.initials: J
authors.lastname: Fourment
authors.forename: C
authors.initials: C
authors.lastname: Ebel
authors.forename: D
authors.initials: D
authors.lastname: Kahn
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D019281:Dimerization; D016296:Mutagenesis; D009586:Nitrogen Fixation; D010766:Phosphorylation; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D016962:Sinorhizobium meliloti / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics; D014461:Ultracentrifugation
chemical_list: D001426:Bacterial Proteins; C061338:NifA protein, Bacteria; D014157:Transcription Factors; C069289:FixJ protein, Bacteria