Heliorhodopsin binds and regulates glutamine synthetase activity.

Photoreceptors are light-sensitive proteins found in various organisms that respond to light and relay signals into the cells. Heliorhodopsin, a retinal-binding membrane protein, has been recently discovered, however its function remains unknown. Herein, we investigated the relationship between Actinobacteria bacterium IMCC26103 heliorhodopsin (AbHeR) and an adjacent glutamine synthetase (AbGS) in the same operon. We demonstrate that AbHeR binds to AbGS and regulates AbGS activity. More specifically, the dissociation constant (Kd) value of the binding between AbHeR and AbGS is 6.06 µM. Moreover, the absence of positively charged residues within the intracellular loop of AbHeR impacted Kd value as they serve as critical binding sites for AbGS. We also confirm that AbHeR up-regulates the biosynthetic enzyme activity of AbGS both in vitro and in vivo in the presence of light. GS is a key enzyme involved in nitrogen assimilation that catalyzes the conversion of glutamate and ammonia to glutamine. Hence, the interaction between AbHeR and AbGS may be critical for nitrogen assimilation in Actinobacteria bacterium IMCC26103 as it survives in low-nutrient environments. Overall, the findings of our study describe, for the first time, to the best of our knowledge, a novel function of heliorhodopsin as a regulatory rhodopsin with the capacity to bind and regulate enzyme activity required for nitrogen assimilation.

Structural basis for the helical filament formation of Escherichia coli glutamine synthetase.

Escherichia coli glutamine synthetase (EcGS) spontaneously forms a dodecamer that catalytically converts glutamate to glutamine. EcGS stacks with other dodecamers to create a filament-like polymer visible under transmission electron microscopy. Filamentous EcGS is induced by environmental metal ions. We used cryo-electron microscopy (cryo-EM) to decipher the structure of metal ion (nickel)-induced EcGS helical filament at a sub-3Å resolution. EcGS filament formation involves stacking of native dodecamers by chelating nickel ions to residues His5 and His13 in the first N-terminal helix (H1). His5 and His13 from paired parallel H1 helices provide salt bridges and hydrogen bonds to tightly stack two dodecamers. One subunit of the EcGS filament hosts two nickel ions, whereas the dodecameric interface and the ATP/Mg-binding site both host a nickel ion each. We reveal that upon adding glutamate or ATP for catalytic reactions, nickel-induced EcGS filament reverts to individual dodecamers. Such tunable filament formation is often associated with stress responses. Our results provide detailed structural information on the mechanism underlying reversible and tunable EcGS filament formation.

Cross-linking mass spectrometry reveals structural insights of the glutamine synthetase from Leishmania braziliensis.

BACKGROUND: Leishmaniasis is a neglected tropical disease caused by the parasite Leishmania braziliensis, commonly found in Brazil and associated with cutaneous and visceral forms of this disease. Like other organisms, L. braziliensis has an enzyme called glutamine synthetase (LbGS) that acts on the synthesis of glutamine from glutamate. This enzyme plays an essential role in the metabolism of these parasites and can be a potential therapeutic target for treating this disease. OBJECTIVES: Investigate LbGS structure and generate structural models of the protein. METHODS: We use the method of crosslinking mass spectrometry (XLMS) and generate structural models in silico using I-TASSER. FINDINGS: 42 XLs peptides were identified, of which 37 are explained in a monomeric model with the other five indicating LbGS dimerization and pentamers interaction region. The comparison of 3D models generated in the presence and absence of XLMS restrictions probed the benefits of modeling with XLMS highlighting the inappropriate folding due to the absence of spatial restrictions. MAIN CONCLUSIONS: In conclusion, we disclose the conservation of the active site and interface regions, but also unique features of LbGS showing the potential of XLMS to probe structural information and explore new drugs.

Assembly status transition offers an avenue for activity modulation of a supramolecular enzyme.

Nature has evolved many supramolecular proteins assembled in certain, sometimes even seemingly oversophisticated, morphological manners. The rationale behind such evolutionary efforts is often poorly understood. Here, we provide atomic-resolution insights into how the dynamic building of a structurally complex enzyme with higher order symmetry offers amenability to intricate regulation. We have established the functional coupling between enzymatic activity and protein morphological states of glutamine synthetase (GS), an old multi-subunit enzyme essential for cellular nitrogen metabolism. Cryo-EM structure determination of GS in both the catalytically active and inactive assembly states allows us to reveal an unanticipated self-assembly-induced disorder-order transition paradigm, in which the remote interactions between two subcomplex entities significantly rigidify the otherwise structurally fluctuating active sites, thereby regulating activity. We further show in vivo evidences that how the enzyme morphology transitions could be modulated by cellular factors on demand. Collectively, our data present an example of how assembly status transition offers an avenue for activity modulation, and sharpens our mechanistic understanding of the complex functional and regulatory properties of supramolecular enzymes.

Crystal structure of N-terminal degron-truncated human glutamine synthetase.

Glutamine synthetase (GS) is a decameric enzyme that plays a key role in nitrogen metabolism. Acetylation of the N-terminal degron (N-degron) of GS is essential for ubiquitylation and subsequent GS degradation. The full-length GS structure showed that the N-degron is buried inside the GS decamer and is inaccessible to the acetyltransferase. The structure of N-degron-truncated GS reported here reveals that the N-degron is not essential for GS decamer formation. It is also shown that the N-degron can be exposed to a solvent region through a series of conformational adjustments upon ligand binding. In summary, this study elucidated the dynamic movement of the N-degron and the possible effect of glutamine in enhancing the acetylation process.

Structural property, molecular regulation, and functional diversity of glutamine synthetase in higher plants: a data-mining bioinformatics approach.

Glutamine synthetase (GS; E.C.6.3.1.2) is a key enzyme in higher plants with two isozymes, cytosolic GS1 and plastidic GS2, and involves in the assimilation and recycling of NH4+ ions and maintenance of complex traits such as crop nitrogen-use efficiency and yield. Our present understanding of crop nitrogen-use efficiency and its correlation with the functional role of the GS family genes is inadequate, which delays harnessing the benefit of this key enzyme in crop improvement. In this report, we performed a comprehensive investigation on the phylogenetic relationship, structural properties, complex multilevel gene regulation, and expression patterns of the GS genes to enrich present understanding about the enzyme. Our Gene Ontology and protein-protein interactions analysis revealed the functional aspects of GS isozymes in stress mitigation, aging, nucleotide biosynthesis/transport, DNA repair and response to metals. The insight gained here contributes to the future research strategies in developing climate-smart crops for global sustainability.

Structural Insight into the Contributions of the N-Terminus and Key Active-Site Residues to the Catalytic Efficiency of Glutamine Synthetase 2.

Glutamine synthetase (GS) catalyzes the condensation of ammonia and glutamate, along with ATP, to form glutamine. Despite extensive studies on GSs from eukaryotes and prokaryotes, the roles of the N-terminus and other structural features in catalysis remain unclear. Here we report the decameric structure of Drosophila melanogaster GS 2 (DmGS2). The N-terminal short helices, α1 and α2, constitute a meander region, and form hydrogen bonds with residues 3-5 in the N-terminal loop, which are not present in the GSs of other species. Deletion of α1 or α1-α2 inactivates DmGS2. Notably, the Arg4 in each monomer of one pentamer forms hydrogen bonds with Glu7, and Asp8 in the adjacent monomer of the other pentamer. Replacement of Arg4 with Asp (R4D) abolishes activity. Analytical ultracentrifugation revealed that Arg4 is crucial for oligomerization. Circular dichroism spectra revealed that R4D may alter the secondary structure. We mutated key residues to identify the substrate-binding site. As Glu140 binds glutamate and Glu311 binds ammonia, mutants E140A and E311A have little activity. Conversely, mutant P214A (P contributes to ATP binding) has higher activity than wild-type DmGS2. These findings expand the understanding of the structural and functional features of the N-terminal meander region of DmGS2 and the residues important for catalytic efficiency.

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.

Adjunctive Immunotherapeutic Efficacy of N-Formylated Internal Peptide of Mycobacterial Glutamine Synthetase in Mouse Model of Tuberculosis.

BACKGROUND: Host-directed therapies are a comparatively new and promising method for the treatment of tuberculosis. A variety of host pathways, vaccines and drugs have the potential to provide novel adjunctive therapies for the treatment of tuberculosis. In this connection, we have earlier reported the immunotherapeutic potential of N-formylated N-terminal peptide of glutamine synthetase of Mycobacterim tuberculosis H37Rv (Mir SA and Sharma S, 2014). Now in the present study, we investigated the immunotherapeutic effect of N-terminally formylated internal-peptide 'f- MLLLPD' of mycobacterial glutamine synthetase (Rv2220) in mouse model of tuberculosis. METHODS: The N-terminally formylated peptide, f-MLLLPD was tested for its potential to generate Reactive Oxygen Species (ROS) in murine neutrophils. Further, its therapeutic effect alone or in combination with anti-tubercular drugs was evaluated in mouse model of tuberculosis. RESULTS: The f-MLLLPD peptide treatment alone and in combination with ATDs reduced the bacterial load (indicated as colony forming units) in lungs of infected mice by 0.58 (p<0.01) and 2.92 (p<0.001) log10 units respectively and in their spleens by 0.46 (p<0.05) and 2.46 (p<0.001) log10 units respectively. In addition, the observed histopathological results correlated well with the CFU data. CONCLUSION: The results of the current study show that f-MLLLPD peptide confers an additional therapeutic efficacy to the anti-tuberculosis drugs.

Structural exploration of glutamine synthetase from Leishmania donovani: Insights from in silico and in vitro analysis.

Glutamine synthetase from L. donovani (LdGS) has been identified as a potential antileishmanial target in our previous report based on biochemical and inhibition studies. With the aim to structurally explore LdGS, systematic in silico and in vitro studies have been employed in the present study to identify amino acids crucial for LdGS mediated catalysis. A comparative analysis with human GS (HsGS) was performed which revealed significant differences in the active site pocket of human and parasite GS enzyme. The important amino acids identified from the in silico analysis of the optimized complexes, were subjected to in silico and in vitro alanine scanning by site directed mutagenesis. The results indicated crucial conserved and non conserved residues required for GS activity. The role of these residues in maintenance of secondary and tertiary structure of GS enzyme was also explored. In silico virtual screening was performed which resulted in the identification of five hits i.e. ZINC83236243, ZINC77319454, ZINC83236244, ZINC83236734 and ZINC83236736, as potential LdGS selective inhibitors. The illustrated structural and functional details of enzyme provides a better understanding of the structural integrity of LdGS and can be further utilized for the development of parasite specific GS inhibitors for treatment of visceral leishmaniasis infections.

A spatial similarity of stereochemical environments formed by amino acid residues defines a common epitope of two non-homologous proteins.

It is critical for development of high-quality antibodies in research and diagnostics to predict accurately their cross-reactivities with "off-target" molecules, which potentially induce false results. Herein, we report a good example of such a cross-reactivity for an off-target due to a stereochemical environment of epitopes, which does not simply depend on amino acid sequences. We found that significant subpopulation of a polyclonal peptide antibody against Bcnt (Bucentaur) (anti-BCNT-C antibody) cross-reacted with a completely different protein, glutamine synthetase (GS), and identified four amino acids, GYFE, in its C-terminal region as the core amino acids for the cross-reaction. Consistent with this finding, the anti-BCNT-C antibody strongly recognized endogenously and exogenously expressed GS in tissues and cultured cells by Western blotting and immunohistochemistry. Furthermore, we elucidated that the cross-reaction is caused by a spatial similarity between the stereochemical environments formed by amino acid residues, including the GYFE of GS and the GYIE of Bcnt, rather than by their primary sequences. These results suggest it is critical to comprehensively analyze antibody interactions with target molecules including off-targets with special attention to the physicochemical environments of epitope-paratope interfaces to decrease the risk of false interpretations of results using antibodies in science and clinical applications.

C-Terminal Part of Glutamate-Ammonia-Ligase Adenyltransferase Gene Identified by RAPD-HRM with 3H Primer for E. Coli Screening.

A single random oligonucleotide 3H primer has been previously applied in random-amplified- polymorphic-DNA (RAPD)-PCR to distinguish stocked bacteria E. coli within a cocktail mixture also containing Enterococcus faecalis, Bifidobacterium longum and Ruminococcus gnavus. In this study, we demonstrate that a 702 base pair (bp) gene fragment can be amplified as a unique pattern by RAPD-PCR using a 3H primer in human faeces containing E. coli. This unique 702 bp amplicon contained a 687 bp gene fragment identified as the C-terminal region of the glutamate-ammonia-ligase adenyltransferase (glnE) gene of E. coli. By high-resolution melt (HRM) analysis, a mean melt-curve temperature of this 702 bp amplicon was determined to be approximately 88.1 ± 0.22 degrees Celsius (°C). A combination of RAPD with HRM in one single reaction based on this amplicon can achieve semi-quantitative detection of up to 102 CFU/ml of E. coli. To increase the signal intensity of HRM, a primer pair capable of screening E. coli directly from fresh human faeces was re-designed from the 687 bp gene segment, giving a mean peak melt-curve temperature at 88.35 ± 0.11 °C. Finally, single-nucleotide polymorphisms of this 687 bp gene segment were analysed for pathogenic E. coli strains, including UMN026, O83:H1, O104:H4, O157:H7 and O169:H41. We conclude that this 687 bp segment of the glnE gene has a high potential for screening of human faecal E. coli, including pathogenic strains, in contaminated food and water.

Functional Research on Three Presumed Asparagine Synthetase Family Members in Poplar.

Asparagine synthetase (AS), a key enzyme in plant nitrogen metabolism, plays an important role in plant nitrogen assimilation and distribution. Asparagine (Asn), the product of asparagine synthetase, is one of the main compounds responsible for organic nitrogen transport and storage in plants. In this study, we performed complementation experiments using an Asn-deficient Escherichia coli strain to demonstrate that three putative asparagine synthetase family members in poplar (Populussimonii× P.nigra) function in Asn synthesis. Quantitative real-time PCR revealed that the three members had high expression levels in different tissues of poplar and were regulated by exogenous nitrogen. PnAS1 and PnAS2 were also affected by diurnal rhythm. Long-term dark treatment resulted in a significant increase in PnAS1 and PnAS3 expression levels. Under long-term light conditions, however, PnAS2 expression decreased significantly in the intermediate region of leaves. Exogenous application of ammonium nitrogen, glutamine, and a glutamine synthetase inhibitor revealed that PnAS3 was more sensitive to exogenous glutamine, while PnAS1 and PnAS2 were more susceptible to exogenous ammonium nitrogen. Our results suggest that the various members of the PnAS gene family have distinct roles in different tissues and are regulated in different ways.

The substrate specificity of aniline dioxygenase is mainly determined by two of its components: glutamine synthetase-like enzyme and oxygenase.

The residues of aniline and its derivatives are serious environment pollutants. Aniline dioxygenase (AD) derived from aerobic bacteria catalyzes the conversion of aniline to catechol, which has potential use in the bioremediation of aromatic amines and biorefining process. AD contains four components: a glutamine synthetase (GS)-like enzyme, a glutamine amidotransferase (GAT)-like enzyme, oxygenase, and reductase. ADs from diverse hosts exhibit different substrate specificities against aniline derivatives. However, what component of AD determines AD's substrate specificity is still unknown which limits the effects of extending AD's substrate spectrum through mutagenesis. Here, each component of two ADs (AtdA1A2A3A4A5 and AdoQTA1A2B) which have different substrate ranges was heterologously expressed and purified. The activity of both ADs was successfully constructed in vitro using the purified components. To identify the component that affects the substrate specificity of the ADs, the substrate specificity of each component was studied. The inability of AtdA1A2A3A4A5 to catalyze 4-methylaniline was determined with GS-like enzyme AtdA1; its inability to convert 2-isopropylaniline was caused by the oxygenase component, and its inability to convert 4-isopropylaniline was caused by both GS-like enzyme AtdA1 and oxygenase components. The inability of AdoQTA1A2B to catalyze 2-methylaniline was determined by GS-like enzyme AdoQ; its inability to convert 2-isopropylaniline was caused by both GS-like enzyme AdoQ and oxygenase components. Together, these results show that GS-like enzyme and oxygenase but not GAT-like enzyme or reductase play dominant roles in the substrate specificity of AD, and this finding will facilitate the engineering of AD to expand its substrate range.

Characterization of plant glutamine synthetase S-nitrosation.

The identification of S-nitrosated substrates and their target cysteine residues is a crucial step to understand the signaling functions of nitric oxide (NO) inside the cells. Here, we show that the key nitrogen metabolic enzyme glutamine synthetase (GS) is a S-nitrosation target in Medicago truncatula and characterize the molecular determinants and the effects of this NO-induced modification on different GS isoenzymes. We found that all the four M. truncatula GS isoforms are S-nitrosated, but despite the high percentage of amino acid identity between the four proteins, S-nitrosation only affects the activity of the plastid-located enzymes, leading to inactivation. A biotin-switch/mass spectrometry approach revealed that cytosolic and plastid-located GSs share an S-nitrosation site at a conserved cysteine residue, but the plastidic enzymes contain additional S-nitrosation sites at non-conserved cysteines, which are accountable for enzyme inactivation. By site-directed mutagenesis, we identified Cys369 as the regulatory S-nitrosation site relevant for the catalytic function of the plastid-located GS and an analysis of the structural environment of the SNO-targeted cysteines in cytosolic and plastid-located isoenzymes explains their differential regulation by S-nitrosation and elucidates the mechanistic by which S-nitrosation of Cys369 leads to enzyme inactivation. We also provide evidence that both the cytosolic and plastid-located GSs are endogenously S-nitrosated in leaves and root nodules of M. truncatula, supporting a physiological meaning for S-nitrosation. Taken together, these results provide new insights into the molecular details of the differential regulation of individual GS isoenzymes by NO-derived molecules and open new paths to explore the biological significance of the NO-mediated regulation of this essential metabolic enzyme.

A glutamine riboswitch is a key element for the regulation of glutamine synthetase in cyanobacteria.

As the key enzyme of bacterial nitrogen assimilation, glutamine synthetase (GS) is tightly regulated. In cyanobacteria, GS activity is controlled by the interaction with inactivating protein factors IF7 and IF17 encoded by the genes gifA and gifB, respectively. We show that a glutamine-binding aptamer within the gifB 5' UTR of Synechocystis sp. PCC 6803 is critical for the expression of IF17. Binding of glutamine induced structural re-arrangements in this RNA element leading to enhanced protein synthesis in vivo and characterizing it as a riboswitch. Mutagenesis showed the riboswitch mechanism to contribute at least as much to the control of gene expression as the promoter-mediated transcriptional regulation. We suggest this and a structurally related but distinct element, to be designated type 1 and type 2 glutamine riboswitches. Extended biocomputational searches revealed that glutamine riboswitches are exclusively but frequently found in cyanobacterial genomes, where they are primarily associated with gifB homologs. Hence, this RNA-based sensing mechanism is common in cyanobacteria and establishes a regulatory feedback loop that couples the IF17-mediated GS inactivation to the intracellular glutamine levels. Together with the previously described sRNA NsiR4, these results show that non-coding RNA is an indispensable component in the control of nitrogen assimilation in cyanobacteria.

Structural Analysis of Glutamine Synthetase from Helicobacter pylori.

Glutamine synthetase (GS) is an enzyme that regulates nitrogen metabolism and synthesizes glutamine via glutamate, ATP, and ammonia. GS is a homo-oligomeric protein of eight, ten, or twelve subunits, and each subunit-subunit interface has its own active site. GS can be divided into GS I, GS II, and GS III. GS I and GS III form dodecamer in bacteria and archaea, whereas GS II form decamer in eukaryotes. GS I can be further subdivided into GS I-α and GS I-ß according to its sequence and regulatory mechanism. GS is an essential protein for the survival of Helicobacter pylori which its infection could promote gastroduodenal diseases. Here, we determined the crystal structures of the GS from H. pylori (Hpy GS) in its apo- and substrate-bound forms at 2.8 Å and 2.9 Å resolution, respectively. Hpy GS formed a dodecamer composed of two hexameric rings stacked face-to-face. Hpy GS, which belongs to GS I, cannot be clearly classified as either GS I-α or GS I-ß based on its sequence and regulatory mechanism. In this study, we propose that Hpy GS could be classified as a new GS-I subfamily and provide structural information on the apo- and substrate-bound forms of the protein.

Quercetin 3-O-glucoside recovered from the wild Egyptian Sahara plant, Euphorbia paralias L., inhibits glutamine synthetase and has antimycobacterial activity.

Tuberculosis remains a major health problem accentuated by the rise of resistance to all available drugs. Therefore, this study was launched to discover a novel antituberculosis agent from wild Egyptian Sahara plants. Twelve such plants were screened, in vitro, for their activity against various Mycobacterium species. The most active plant, Euphorbia paralias, was further fractionated with different organic solvents, and the activity of the obtained fractions was determined by the agar diffusion and broth microdilution methods. The methanol fraction was the most active against Mycobacterium spp., and was non-toxic in doses up to 10 g/kg of animal weight. Its main component was separated by column chromatography, and then identified by ultraviolet spectroscopy and nuclear magnetic resonance analysis as quercetin-3-O-ß-D-glucoside. Docking analysis suggested that quercetin-3-O-ß-D-glucoside inhibits the glutamine synthetase enzyme, a promising target for the development of antituberculosis drugs. This prediction was confirmed by an in vitro glutamine synthetase biosynthetic assay. To the best of our knowledge, and based on bioinformatics mining of the BioPhytMol database, this is the first report on the antimycobacterial activity of Euphorbia paralias plant. It is also the first report on the inhibition of mycobacterial glutamine synthetase by the flavonoid quercetin.

Short- and long-term effects on mAb-producing CHO cell lines after cryopreservation.

Cryopreservation provides the foundation for research, development, and manufacturing operations in the CHO-based biopharmaceutical industry. Despite its criticality, studies are lacking that explicitly demonstrate that the routine cell banking process and the potential stress and damage during cryopreservation and recovery from thaw have no lasting detrimental effects on CHO cells. Statistics are also scarce on the decline of cell-specific productivity (Qp ) over time for recombinant CHO cells developed using the glutamine synthetase (GS)-based methionine sulfoximine (MSX) selection system. To address these gaps, we evaluated the impact of freeze-thaw on 24 recombinant CHO cell lines (generated by the GS/MSX selection system) using a series of production culture assays. Across the panel of cell lines expressing one of three monoclonal antibodies (mAbs), freeze-thaw did not result in any significant impact beyond the initial post-thaw passages. Production cultures sourced from cryopreserved cells and their non-cryopreserved counterparts yielded similar performance (growth, viability, and productivity), product quality (size, charge, and glycosylation distributions), and flow cytometric profiles (intracellular mAb expression). However, many production cultures yielded lower Qp at increased cell age: 17 of the 24 cell lines displayed ≥20% Qp decline after ∼2-3 months of passaging, irrespective of whether the cells were previously cryopreserved. The frequency of Qp decline underscores the continued need for understanding the underlying mechanisms and for careful clone selection. Because our experiments were designed to decouple the effects of cryopreservation from those of cell age, we could conclusively rule out freeze-thaw as a cause for Qp decline. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:463-477, 2018.

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 0.4µM, 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.