title: Dissecting the Binding between Glutamine Synthetase and Its Two Natively Unfolded Protein Inhibitors.
abstract: Ammonium is incorporated into carbon skeletons by the sequential action of glutamine synthetase (GS) and glutamate synthase (GOGAT) in cyanobacteria. The activity of Synechocystis sp. PCC 6803 GS type I is controlled by protein-protein interactions with two intrinsically disordered inactivating factors (IFs): the 65-residue (IF7) and the 149-residue one (IF17). In this work, we studied both IF7 and IF17 by nuclear magnetic resonance (NMR), and we described their binding to GS by using NMR and biolayer interferometry. We assigned the backbone nuclei of all residues of IF7. Analyses of chemical shifts and the (15)N-{(1)H} NOEs at two field strengths suggest that IF7 region Thr3-Arg13 and a few residues around Ser27 and Phe41 populated helical conformations (although the percentage is smaller around Phe41). The two-dimensional (1)H-(15)N HSQC and CON experiments suggest that IF17 populated several conformations. We followed the binding between GS and IF7 by NMR at physiological pH, and the residues interacting first with IF7 were Gln6 and Ser27, belonging to those regions that appeared to be ordered in the isolated protein. We also determined the kon values and koff values for the binding of both IF7 and IF17 to GS, where the GS protein was bound to a biosensor. The measurements of the kinetic constants for the binding of IF7 to GS suggest that: (i) binding does not follow a kinetic two-state model ([Formula: see text]), (ii) there is a strong electrostatic component in the determined kon, and (iii) the binding is not diffusion-limited.
authors.affiliation: Instituto de Química Física Rocasolano (IQFR), CSIC , 28006 Madrid, Spain.
authors.forename: David
authors.initials: D
authors.lastname: Pantoja-Uceda
authors.affiliation: Instituto de Biología Molecular y Celular, Universidad Miguel Hernández , 03202 Elche (Alicante), Spain.|Instituto de Biocomputación y Física de Sistemas Complejos (BIFI), Unidad Asociada IQFR-CSIC-BIFI, Universidad de Zaragoza , 50009 Zaragoza, Spain.
authors.forename: José L
authors.initials: JL
authors.lastname: Neira
authors.affiliation: Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC-Universidad de Sevilla , 41092 Seville, Spain.
authors.forename: Lorena
authors.initials: L
authors.lastname: Saelices
authors.affiliation: Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC-Universidad de Sevilla , 41092 Seville, Spain.
authors.forename: Rocío
authors.initials: R
authors.lastname: Robles-Rengel
authors.affiliation: Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC-Universidad de Sevilla , 41092 Seville, Spain.
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Florencio
authors.affiliation: Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC-Universidad de Sevilla , 41092 Seville, Spain.
authors.forename: M Isabel
authors.initials: MI
authors.lastname: Muro-Pastor
authors.affiliation: Instituto de Química Física Rocasolano (IQFR), CSIC , 28006 Madrid, Spain.
authors.forename: Jorge
authors.initials: J
authors.lastname: Santoro
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D002942:Circular Dichroism; D015964:Gene Expression Regulation, Bacterial; D005974:Glutamate-Ammonia Ligase / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D046939:Synechocystis / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; C120035:IF17 protein, Synechocystis sp.; C120034:IF7 protein, Synechocystis sp.; D011994:Recombinant Proteins; D005974:Glutamate-Ammonia Ligase