caa a22 4500 605507929 CHVBK 20210128100634.0 cr unu---uuuuu 210128e20150301xx s 000 0 eng 10.1007/s00775-014-1232-4 doi (NATIONALLICENCE)springer-10.1007/s00775-014-1232-4 Dual binding of 14-3-3 protein regulates Arabidopsis nitrate reductase activity [Elektronische Daten] [Jen-Chih Chi, Juliane Roeper, Guenter Schwarz, Katrin Fischer-Schrader] 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. SBIC, 2015 Phosphorylation nationallicence Acidic motif nationallicence 14-3-3 nationallicence Nitrate reductase nationallicence Regulation nationallicence EDC : 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride nationallicence GST : Glutathione-S-transferase nationallicence NR : Nitrate reductase nationallicence NT : N-terminus of nitrate reductase nationallicence SPR : Surface plasmon resonance spectroscopy nationallicence wt : Wild-type nationallicence Chi Jen-Chih Department of Chemistry, Institute for Biochemistry, University of Cologne, 50674, Cologne, Germany aut Roeper Juliane Department of Chemistry, Institute for Biochemistry, University of Cologne, 50674, Cologne, Germany aut Schwarz Guenter Department of Chemistry, Institute for Biochemistry, University of Cologne, 50674, Cologne, Germany aut Fischer-Schrader Katrin Department of Chemistry, Institute for Biochemistry, University of Cologne, 50674, Cologne, Germany aut JBIC Journal of Biological Inorganic Chemistry Springer Berlin Heidelberg 20/2(2015-03-01), 277-286 0949-8257 20:2<277 2015 20 775 https://doi.org/10.1007/s00775-014-1232-4 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00775-014-1232-4 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Chi Jen-Chih Department of Chemistry, Institute for Biochemistry, University of Cologne, 50674, Cologne, Germany aut NATIONALLICENCE 700 1- Roeper Juliane Department of Chemistry, Institute for Biochemistry, University of Cologne, 50674, Cologne, Germany aut NATIONALLICENCE 700 1- Schwarz Guenter Department of Chemistry, Institute for Biochemistry, University of Cologne, 50674, Cologne, Germany aut NATIONALLICENCE 700 1- Fischer-Schrader Katrin Department of Chemistry, Institute for Biochemistry, University of Cologne, 50674, Cologne, Germany aut NATIONALLICENCE 773 0- JBIC Journal of Biological Inorganic Chemistry Springer Berlin Heidelberg 20/2(2015-03-01), 277-286 0949-8257 20:2<277 2015 20 775