Phosphorylation-induced dimerization of the FixJ receiver domain

Authors

  • Sandra Da Re,

    1. Laboratoire de Biologie Moléculaire des Relations Plantes-Microorganismes, UMR 215 INRA-CNRS, Chemin de Borde Rouge, BP 27, 31326 Castanet-Tolosan Cedex, France.,
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    • Jörg Schumacher,

      1. Laboratoire de Biologie Moléculaire des Relations Plantes-Microorganismes, UMR 215 INRA-CNRS, Chemin de Borde Rouge, BP 27, 31326 Castanet-Tolosan Cedex, France.,
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      • Philippe Rousseau,

        1. Laboratoire de Biologie Moléculaire des Relations Plantes-Microorganismes, UMR 215 INRA-CNRS, Chemin de Borde Rouge, BP 27, 31326 Castanet-Tolosan Cedex, France.,
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      • Joëlle Fourment,

        1. Laboratoire de Biologie Moléculaire des Relations Plantes-Microorganismes, UMR 215 INRA-CNRS, Chemin de Borde Rouge, BP 27, 31326 Castanet-Tolosan Cedex, France.,
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      • Christine Ebel,

        1. Institut de Biologie Structurale Jean-Pierre Ebel, UPR 9015 CNRS, CEA, 41, rue Jules Horowitz, 38027 Grenoble Cedex 1, France.
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      • Daniel Kahn

        1. Laboratoire de Biologie Moléculaire des Relations Plantes-Microorganismes, UMR 215 INRA-CNRS, Chemin de Borde Rouge, BP 27, 31326 Castanet-Tolosan Cedex, France.,
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      Daniel Kahn. E-mail dkahn@toulouse.inra.fr; Tel. (+33) 561 28 53 29; Fax (+33) 561 28 50 61.

      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 ≈ 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~P dimerization interface involves Val-91 and Lys-95 in helix α4. Dimerization was required for high-affinity binding to fixK promoter DNA.

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