Universität zu Köln

Chi, Jen-Chih, Roeper, Juliane, Schwarz, Guenter ORCID: 0000-0002-2118-9338 and Fischer-Schrader, Katrin ORCID: 0000-0003-4532-8918 (2015). Dual binding of 14-3-3 protein regulates Arabidopsis nitrate reductase activity. J. Biol. Inorg. Chem., 20 (2). S. 277 - 287. NEW YORK: SPRINGER. ISSN 1432-1327

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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 14-3-3 omega to the well-known phospho-hinge site and low-affinity binding to the N-terminal acidic motif. A binding groove-deficient 14-3-3 omega 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 14-3-3 omega to confer full inhibition of NR activity under physiological conditions.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Chi, Jen-Chih UNSPECIFIED UNSPECIFIED UNSPECIFIED Roeper, Juliane UNSPECIFIED UNSPECIFIED UNSPECIFIED Schwarz, Guenter UNSPECIFIED orcid.org/0000-0002-2118-9338 UNSPECIFIED Fischer-Schrader, Katrin UNSPECIFIED orcid.org/0000-0003-4532-8918 UNSPECIFIED
URN:	urn:nbn:de:hbz:38-406300
DOI:	10.1007/s00775-014-1232-4
Journal or Publication Title:	J. Biol. Inorg. Chem.
Volume:	20
Number:	2
Page Range:	S. 277 - 287
Date:	2015
Publisher:	SPRINGER
Place of Publication:	NEW YORK
ISSN:	1432-1327
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Faculty of Mathematics and Natural Sciences > Department of Chemistry > Institute of Biochemistry
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language N-TERMINAL DOMAIN; HINGE 1 REGION; PHOSPHORYLATION SITE; FUNCTIONAL SPECIFICITY; EXOENZYME-S; IN-VIVO; IDENTIFICATION; ACTIVATION; ENZYME; MECHANISM Multiple languages Biochemistry & Molecular Biology; Chemistry, Inorganic & Nuclear Multiple languages
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/40630