title: Dual binding of 14-3-3 protein regulates Arabidopsis nitrate reductase activity.
abstract: 14-3-3 proteins represent a family of ubiquitous eukaryotic proteins involved in numerous signal transduction processes and metabolic pathways. One important 14-3-3 target in higher plants is nitrate reductase (NR), whose activity is regulated by different physiological conditions. Intra-molecular electron transfer in NR is inhibited following 14-3-3 binding to a conserved phospho-serine motif located in hinge 1, a surface exposed loop between the catalytic molybdenum and central heme domain. Here we describe a novel 14-3-3 binding site within the NR N-terminus, an acidic motif conserved in NRs of higher plants, which significantly contributes to 14-3-3-mediated inhibition of NR. Deletion or mutation of the N-terminal acidic motif resulted in a significant loss of 14-3-3 mediated inhibition of Ser534 phosphorylated NR-Mo-heme (residues 1-625), a previously established model of NR regulation. Co-sedimentation and crosslinking studies with NR peptides comprising each of the two binding motifs demonstrated direct binding of either peptide to 14-3-3. Surface plasmon resonance spectroscopy disclosed high-affinity binding of 14-3-3ω to the well-known phospho-hinge site and low-affinity binding to the N-terminal acidic motif. A binding groove-deficient 14-3-3ω variant retained interaction to the acidic motif, but lost binding to the phospho-hinge motif. To our knowledge, NR is the first enzyme that harbors two independent 14-3-3 binding sites with different affinities, which both need to be occupied by 14-3-3ω to confer full inhibition of NR activity under physiological conditions.
authors.affiliation: Department of Chemistry, Institute for Biochemistry, University of Cologne, 50674, Cologne, Germany.
authors.forename: Jen-Chih
authors.initials: JC
authors.lastname: Chi
authors.forename: Juliane
authors.initials: J
authors.lastname: Roeper
authors.forename: Guenter
authors.initials: G
authors.lastname: Schwarz
authors.forename: Katrin
authors.initials: K
authors.lastname: Fischer-Schrader
mesh_terms: D048948:14-3-3 Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D004579:Electron Transport; D009154:Mutation; D050901:Nitrate Reductase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid
chemical_list: D048948:14-3-3 Proteins; D050901:Nitrate Reductase