A core of three amino acids at the carboxyl-terminal region of glutamine synthetase defines its regulation in cyanobacteria

© 2015 John Wiley & Sons Ltd. Glutamine synthetase (GS) type I is a key enzyme in nitrogen metabolism, and its activity is finely controlled by cellular carbon/nitrogen balance. In cyanobacteria, a reversible process that involves protein-protein interaction with two proteins, the inactivating factors IF7 and IF17, regulates GS. Previously, we showed that three arginine residues of IFs are critical for binding and inhibition of GS. In this work, taking advantage of the specificity of GS/IFs interaction in the model cyanobacteria Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120, we have constructed a different chimeric GSs from these two cyanobacteria. Analysis of these proteins, together with a site-directed mutagenesis approach, indicates that a core of three residues (E419, N456 and R459) is essential for the inactivation process. The three residues belong to the last 56 amino acids of the C-terminus of SynechocystisGS. A protein-protein docking modeling of SynechocystisGS in complex with IF7 supports the role of the identified core for GS/IF interaction.Peer Reviewe

Dynamics of the Intrinsically Disordered Inhibitor IF7 of Glutamine Synthetase in Isolation and in Complex with its Partner

Glutamine synthetase (GS) catalyzes the ATP-dependent formation of glutamine from glutamate and ammonia. The activity of Synechocystis sp. PCC 6803 GS is regulated, among other mechanisms, by protein-protein interactions with a 65-residue-long, intrinsically disordered protein (IDP), named IF7. IDPs explore diverse conformations in their free states and, in some cases, in their molecular complexes. We used both nuclear magnetic resonance (NMR) at 11.7 T and small angle X-ray scattering (SAXS) to study the size and the dynamics in the picoseconds-to-nanosecond (ps-ns) timescale of: (i) isolated IF7; and (ii) the IF7/GS complex. Our SAXS findings, together with MD results, show: (i) some of the possible IF7 structures in solution; and, (ii) that the presence of IF7 affected the structure of GS in solution. The joint use of SAXS and NMR shows that movements of each amino acid of IF7 were uncorrelated with those of its neighbors. Residues of IF7 with the largest values of the relaxation rates (R1, R2 and ηxy), in the free and bound species, were mainly clustered around: (i) the C terminus of the protein; and (ii) Ala30. These residues, together with Arg8 (which is a hot-spot residue in the interaction with GS), had a restricted mobility in the presence of GS. The C-terminal region, which appeared more compact in our MD simulations of isolated IF7, seemed to be involved in non-native contacts with GS that help in the binding between the two macromolecules.Ministerio de Economía y Competitividad RTI 2018-097991- BI0

The cyanobacterial ribosomal-associated protein LrtA from Synechocystis sp. PCC 6803 is an oligomeric protein in solution with chameleonic sequence properties

The LrtA protein of Synechocystis sp. PCC 6803 intervenes in cyanobacterial post-stress survival and in stabilizing 70S ribosomal particles. It belongs to the hibernating promoting factor (HPF) family of proteins, involved in protein synthesis. In this work, we studied the conformational preferences and stability of isolated LrtA in solution. At physiological conditions, as shown by hydrodynamic techniques, LrtA was involved in a self-association equilibrium. As indicated by Nuclear Magnetic Resonance (NMR), circular dichroism (CD) and fluorescence, the protein acquired a folded, native-like conformation between pH 6.0 and 9.0. However, that conformation was not very stable, as suggested by thermal and chemical denaturations followed by CD and fluorescence. Theoretical studies of its highly-charged sequence suggest that LrtA had a Janus sequence, with a context-dependent fold. Our modelling and molecular dynamics (MD) simulations indicate that the protein adopted the same fold observed in other members of the HPF family ( - - - - - ) at its N-terminal region (residues 1–100), whereas the C terminus (residues 100–197) appeared disordered and collapsed, supporting the overall percentage of overall secondary structure obtained by CD deconvolution. Then, LrtA has a chameleonic sequence and it is the first member of the HPF family involved in a self-association equilibrium, when isolated in solution.Ministerio de Economía y Competitividad CTQ2015-64445-RMinisterio de Economía y Competitividad BIO2016-78020-RMinisterio de Economía y Competitividad FIS2014-52212-RMinisterio de Economía y Competitividad BIO2016-75634-PFundación Séneca 19353/PI/1

The isolated, twenty-three-residue-long, N-terminal region of the glutamine synthetase inactivating factor binds to its target

Glutamine synthetase (GS) catalyzes the ATP-dependent formation of glutamine from glutamate and ammonia. The activity of Synechocystis sp. PCC 6803 GS type I is regulated by protein-protein interactions with a 65-residue-long protein (IF7). IF7 binds initially to GS through residues at its N terminus. In this work, we studied the conformational preferences of the N-terminal region of IF7 (IF7pep, residues Ala7-Ala29), its binding to GS and its functional properties. Isolated IF7pep populated a nascent helix in aqueous solution. IF7pep was bound to GS with an affinity constant of 0.4 μM, and a 1:1 stoichiometry. IF7pep did not inactivate GS, suggesting that there were other IF7 regions important to carry out the inactivating function. Binding of IF7pep to GS was electrostatically-driven and it did not follow a kinetic two-state model.Peer Reviewe

The Cyanobacterial Ribosomal-Associated Protein LrtA Is Involved in Post-Stress Survival in Synechocystis sp. PCC 6803.

A light-repressed transcript encodes the LrtA protein in cyanobacteria. We show that half-life of lrtA transcript from Synechocystis sp. PCC 6803 is higher in dark-treated cells as compared to light-grown cells, suggesting post-transcriptional control of lrtA expression. The lrtA 5´ untranslated leader region is involved in that darkness-dependent regulation. We also found that Synechocystis sp. PCC 6803 LrtA is a ribosome-associated protein present in both 30S and 70S ribosomal particles. In order to investigate the function of this protein we have constructed a deletion mutant of the lrtA gene. Cells lacking LrtA (∆lrtA) had significantly lower amount of 70S particles and a greater amount of 30S and 50S particles, suggesting a role of LrtA in stabilizing 70S particles. Synechocystis strains with different amounts of LrtA protein: wild-type, ∆lrtA, and LrtAS (overexpressing lrtA) showed no differences in their growth rate under standard laboratory conditions. However, a clear LrtA dose-dependent effect was observed in the presence of the antibiotic tylosin, being the LrtAS strains the most sensitive. Similar results were obtained under hyperosmotic stress caused by sorbitol. Conversely, after prolonged periods of starvation, ∆lrtA strains were delayed in their growth with respect to the wild-type and the LrtAS strains. A positive role of LrtA protein in post-stress survival is proposed