Dissecting the Binding between Glutamine Synthetase and Its Two Natively Unfolded Protein Inhibitors.

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-long (IF7) and the 149-residue-long (IF17). In this work, we studied both

the IF7 and IF17 by Nuclear Magnetic Resonance (NMR), and we described their binding to GS, by

using NMR and biolayer interferometry (BLI). We assigned the backbone nuclei of all residues of IF7.

Analysis of chemical shifts and the 15N-{1H}-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 2D 1H-15N 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 firstly with IF7 were Gly34, Lys39 and Phe41 (and/or

Trp42), close to those regions that appeared ordered. We also determined the kons and koffs for the

binding reactions to GS of both IF7 and IF17, where the GS protein was bound to a biosensor. The

measurements of the kinetic constants for the binding reaction of IF7 to GS suggest that: (i) binding

does not follow a kinetic two-state model ; (ii) there is a strong

electrostatic component in the apparent kon; and, (iii) the binding is not diffusion-limited.