The PII protein interacts with the Amt ammonium transport and modulates nitrate/nitrite assimilation in mycobacteria.

PII proteins are signal transduction proteins that belong to a widely distributed family of proteins involved in the modulation of different metabolisms in bacteria. These proteins are homotrimers carrying a flexible loop, named T-loop, which changes its conformation due to the recognition of diverse key metabolites, ADP, ATP, and 2-oxoglutarate. PII proteins interact with different partners to primarily regulate a set of nitrogen pathways. In some organisms, PII proteins can also control carbon metabolism by interacting with the biotin carboxyl carrier protein (BCCP), a key component of the acetyl-CoA carboxylase (ACC) enzyme complex, inhibiting its activity with the consequent reduction of fatty acid biosynthesis. Most bacteria contain at least two PII proteins, named GlnB and GlnK, with different regulatory roles. In mycobacteria, only one PII protein was identified, and the three-dimensional structure was solved, however, its physiological role is unknown. In this study we purified the Mycobacterium tuberculosis (M. tb) PII protein, named GlnB, and showed that it weakly interacts with the AccA3 protein, the α subunit shared by the three different, and essential, Acyl-CoA carboxylase complexes (ACCase 4, 5, and 6) present in M. tb. A M. smegmatis deletion mutant, ∆MsPII, exhibited a growth deficiency on nitrate and nitrite as unique nitrogen sources, and accumulated nitrite in the culture supernatant. In addition, M. tb PII protein was able to interact with the C-terminal domain of the ammonium transporter Amt establishing the ancestral role for this PII protein as a GlnK functioning protein.

A simple preparation of prestained molecular markers for electrophoresis using inexpensive and readily available proteins.

Protein electrophoresis in polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS-PAGE) is one of the most commonly performed procedures in biochemical laboratories. It requires the use of molecular weight (MW) markers as an internal technical control and to determine the migration rate of a particular protein. In this work, we describe a simple method for preparing "homemade" prestained protein markers using readily available cow's milk and chicken egg white proteins without the need of any major protein purification step, and produce prestained MW markers ranging from 19 to 98 kDa.

Expression, purification and characterization of the transcription termination factor Rho from Azospirillum brasilense.

The Transcription Termination factor Rho is a ring-shaped, homohexameric protein that causes transcript termination by interaction with specific sites on nascent mRNAs. The process of transcription termination is essential for proper expression and regulation of bacterial genes. Although Rho has been extensively studied in the model bacteria Escherichia coli (EcRho), the properties of other Rho orthologues in other bacteria are poorly characterized. Here we present the heterologous expression and purification of untagged Rho protein from the diazotrophic environmental bacterium Azospirillum brasilense (AbRho). The AbRho protein was purified to >99% through a simple, reproducible and efficient purification protocol, a two-step chromatography procedure (affinity/gel filtration). By using analytical gel filtration and dynamic light scattering (DLS), we found that AbRho is arranged as an homohexamer as observed in the EcRho orthologue. Secondary structure and enzyme activity of AbRho was also evaluate indicating a properly folded and active protein after purification. Enzymatic assays indicate that AbRho is a RNA-dependent NTPase enzyme.

The microbiome of a shell mound: ancient anthropogenic waste as a source of Streptomyces degrading recalcitrant polysaccharides.

Metagenome amplicon DNA sequencing and traditional cell culture techniques are helping to uncover the diversity and the biotechnological potential of prokaryotes in different habitats around the world. It has also had a profound impact on microbial taxonomy in the last decades. Here we used metagenome 16S rDNA amplicon sequencing to reveal the microbiome composition of different layers of an anthropogenic soil collected at a shell mound Sambaqui archeological site. The Samabaqui soil microbiome is mainly composed by phyla Acidobacteria, Rokubacteria, Proteobacteria and Thaumarchaeota. Using culture-dependent analysis we obtained few Streptomyces isolates from the Sambaqui soil. One of the isolates, named Streptomyces sp. S3, was able to grow in minimal medium containing recalcitrant polysaccharides including chitin, xylan, carboxymethylcellulose or microcrystalline cellulose as sole carbon sources. The activities of enzymes degrading these compounds were confirmed in cell free supernatants. The genome sequence revealed not only an arsenal of genes related to polysaccharides degradation but also biosynthetic gene clusters which may be involved in the production of biotechnologically interesting secondary metabolites.

The protein-protein interaction network of the Escherichia coli EIIANtr regulatory protein reveals a role in cell motility and metabolic control.

The nitrogen-related PTSNtr system, present in many Proteobacteria including Escherichia coli, acts as a phosphorelay cascade composed of the EINtr, NPr and EIIANtr proteins. Phosphotransfer initiates with phosphoenolpyruvate-dependent EINtr autophosphorylation, the phosphoryl group is then transferred to NPr and finally to a conserved histidine residue on EIIANtr. The reporter metabolites l-glutamine and 2-oxoglutarate reciprocally regulate EINtr autophosphorylation (Lee et al., 2013) and consequently the phosphorylation status of the PTSNtr components is controlled by the availability of nitrogen and carbon. The final phosphate acceptor, EIIANtr, regulates a range of cellular process by acting as the central hub of a complex protein-protein interaction network. Contact between EIIANtr and its target proteins is usually regulated by the EIIANtr phosphorylation status. In this study we performed ligand fishing assays coupled to label-free quantitative proteomics to examine the protein-protein interaction network of E. coli EIIANtr and a phosphomimic variant of the protein. The ligand fishing data, along with phenotypic analysis, indicated that EIIANtr interacts with proteins related to chemotaxis and thereby regulates cell motility. Important metabolic enzymes were also identified as potential EIIANtr binding partners.

Magnetic Bead-Based Immunoassay Allows Rapid, Inexpensive, and Quantitative Detection of Human SARS-CoV-2 Antibodies.

Immunological methods to detect SARS-CoV-2 seroconversion in humans are important to track COVID-19 cases and the humoral response to SARS-CoV-2 infections and immunization to future vaccines. The aim of this work was to develop a simple chromogenic magnetic bead-based immunoassay which allows rapid, inexpensive, and quantitative detection of human antibodies against SARS-CoV-2 in serum, plasma, or blood. Recombinant 6xHis-tagged SARS-CoV-2 Nucleocapsid protein was mobilized on the surface of Ni2+ magnetic beads and challenged with serum or blood samples obtained from controls or COVID-19 cases. The beads were washed, incubated with anti-human IgG-HPR conjugate, and immersed into a solution containing a chromogenic HPR substrate. Bead transfer and homogenization between solutions was aided by a simple low-cost device. The method was validated by two independent laboratories, and the performance to detect SARS-CoV-2 seroconversion in humans was in the same range as obtained using the gold standard immunoassays ELISA and Luminex, though requiring only a fraction of consumables, instrumentation, time to deliver results, and volume of sample. Furthermore, the results obtained with the method described can be visually interpreted without compromising accuracy as demonstrated by validation at a point-of-care unit. The magnetic bead immunoassay throughput can be customized on demand and is readily adapted to be used with any other 6xHis tagged protein or peptide as antigen to track other diseases.

The Protein-Protein Interaction Network Reveals a Novel Role of the Signal Transduction Protein PII in the Control of c-di-GMP Homeostasis in Azospirillum brasilense.

The PII family comprises a group of widely distributed signal transduction proteins ubiquitous in prokaryotes and in the chloroplasts of plants. PII proteins sense the levels of key metabolites ATP, ADP, and 2-oxoglutarate, which affect the PII protein structure and thereby the ability of PII to interact with a range of target proteins. Here, we performed multiple ligand fishing assays with the PII protein orthologue GlnZ from the plant growth-promoting nitrogen-fixing bacterium Azospirillum brasilense to identify 37 proteins that are likely to be part of the PII protein-protein interaction network. Among the PII targets identified were enzymes related to nitrogen and fatty acid metabolism, signaling, coenzyme synthesis, RNA catabolism, and transcription. Direct binary PII-target complex was confirmed for 15 protein complexes using pulldown assays with recombinant proteins. Untargeted metabolome analysis showed that PII is required for proper homeostasis of important metabolites. Two enzymes involved in c-di-GMP metabolism were among the identified PII targets. A PII-deficient strain showed reduced c-di-GMP levels and altered aerotaxis and flocculation behavior. These data support that PII acts as a major metabolic hub controlling important enzymes and the homeostasis of key metabolites such as c-di-GMP in response to the prevailing nutritional status.IMPORTANCE The PII proteins sense and integrate important metabolic signals which reflect the cellular nutrition and energy status. Such extraordinary ability was capitalized by nature in such a way that the various PII proteins regulate different facets of metabolism by controlling the activity of a range of target proteins by protein-protein interactions. Here, we determined the PII protein interaction network in the plant growth-promoting nitrogen-fixing bacterium Azospirillum brasilense The interactome data along with metabolome analysis suggest that PII functions as a master metabolic regulator hub. We provide evidence that PII proteins act to regulate c-di-GMP levels in vivo and cell motility and adherence behaviors.

The NADP-dependent malic enzyme MaeB is a central metabolic hub controlled by the acetyl-CoA to CoASH ratio.

Malic enzymes participate in key metabolic processes, the MaeB-like malic enzymes carry a catalytic inactive phosphotransacetylase domain whose function remains elusive. Here we show that acetyl-CoA directly binds and inhibits MaeB-like enzymes with a saturable profile under physiological relevant acetyl-CoA concentrations. A MaeB-like enzyme from the nitrogen-fixing bacterium Azospirillum brasilense, namely AbMaeB1, binds both acetyl-CoA and unesterified CoASH in a way that inhibition of AbMaeB1 by acetyl-CoA is relieved by increasing CoASH concentrations. Hence, AbMaeB1 senses the acetyl-CoA/CoASH ratio. We revisited E. coli MaeB regulation to determine the inhibitory constant for acetyl-CoA. Our data support that the phosphotransacetylase domain of MaeB-like enzymes senses acetyl-CoA to dictate the fate of carbon distribution at the phosphoenol-pyruvate / pyruvate / oxaloacetate metabolic node.

Production, purification, and characterization of a novel serine-esterase from Aspergillus westerdijkiae.

Esterases hydrolyze water soluble short chain fatty acids esters and are biotechnologically important. A strain of Aspergillus westerdijkiae isolated from cooking oil for recycling was found to secrete an esterase. The best enzyme production (19-24 U/ml of filtrate) culture conditions were stablished. The protein was purified using ammonium sulphate precipitation, dialysis, and a chromatographic step in Sephacryl S-200 HR. The 32 kDa purified protein presented an optimal temperature of 40°C, with a T50 of 48.95°C, and an optimal pH of 8.0. KM and Vmax were 638.11 µM for p-NPB and 5.47 µmol of released p-NP · min-1 · µg-1 of protein, respectively. The purified enzyme was partially active in the presence of 25% acetone. PMSF inhibited the enzyme, indicating that it is a serine hydrolase. MS enzyme peptides sequences were used to find the protein in the A. westerdijkiae sequenced genome. A structure model demonstrated that the protein is a member of the a/ß -hydrolase fold superfamily.

Proteome analysis of an Escherichia coli ptsN-null strain under different nitrogen regimes.

The carbohydrate-uptake phosphorelay PTS system plays a key role in metabolic regulation in Bacteria controlling the utilization of secondary carbon sources. Some bacteria, such as Escherichia coli, encode a paralogous system named PTSNtr (nitrogen related PTS). PTSNtr is composed of EINtr (ptsP), NPr (ptsO), and EIIANtr (ptsN). These proteins act as a phosphorelay system from phosphoenolpyruvate to EINtr, NPr and them to EIIANtr. PTSNtr is not involved in carbohydrate uptake and it may be dedicated to performing regulatory functions. The phosphorylation state of EINtr is regulated by allosteric binding of glutamine and 2-oxoglutarate, metabolites whose intracellular levels reflect the nitrogen status. Although PTSNtr is designated as having nitrogen-sensory properties, no major effect of this system on nitrogen regulation has been described in E. coli. Here we show that an E. coli ptsN deletion mutant has impaired growth in minimal medium. Proteome analysis of the ∆ptsN strain under different nitrogen regimes revealed no involvement in regulation of the canonical nitrogen regulatory (Ntr) system. The proteomic data support the conclusion that ptsN is required to balance the activities of the sigma factors RpoS and RpoD in such way that, in the absence of ptsN, RpoS-dependent genes are preferentially expressed. SIGNIFICANCE: The nitrogen related PTSNtr phosphorelay system has been hypothesized to participate in the control of nitrogen metabolism. Here we used a proteomics approach to show that an Escherichia coli ptsN null strain, which misses the final module of PTSNtr phosphorelay, has no significant effects on nitrogen metabolism under different nitrogen regimes. We noted that ptsN is required for fitness under minimal medium and for the proper balance between RpoS and sigma 70 activities in such way that, in the absence of ptsN, RpoS-dependent genes are preferentially expressed.

Expression and purification of untagged GlnK proteins from actinobacteria.

The PII protein family constitutes one of the most conserved and well distributed family of signal transduction proteins in nature. These proteins play key roles in nitrogen and carbon metabolism. PII function has been well documented in Gram-negative bacteria. However, there are very few reports describing the in vitro properties and function of PII derived from Gram-positive bacteria. Here we present the heterologous expression and efficient purification protocols for untagged PII from three Actinobacteria of medical and biotechnological interest namely: Mycobacterium tuberculosis, Rhodococcus jostii and Streptomyces coelicolor. Circular dichroism and gel filtration analysis supported that the purified proteins are correctly folded. The purification protocol described here will facilitate biochemical studies and help to uncover the biochemical functions of PII proteins in Actinobacteria.

Dynamics of the Escherichia coli proteome in response to nitrogen starvation and entry into the stationary phase.

Nitrogen is needed for the biosynthesis of biomolecules including proteins and nucleic acids. In the absence of fixed nitrogen prokaryotes such as E. coli immediately ceases growth. Ammonium is the preferred nitrogen source for E. coli supporting the fastest growth rates. Under conditions of ammonium limitation, E. coli can use alternative nitrogen sources to supply ammonium ions and this reprogramming is led by the induction of the NtrC regulon. Here we used label free proteomics to determine the dynamics of E. coli proteins expression in response to ammonium starvation in both the short (30min) and the longer (60min) starvation. Protein abundances and post-translational modifications confirmed that activation of the NtrC regulon acts as the first line of defense against nitrogen starvation. The ribosome inactivating protein Rmf was induced shortly after ammonium exhaustion and this was preceded by induction of other ribosome inactivating proteins such as Hpf and RaiA supporting the hypothesis that ribosome shut-down is a key process during nitrogen limitation stress. The proteomic data revealed that growth arrest due to nitrogen starvation correlates with the accumulation of proteins involved in DNA condensation, RNA and protein catabolism and ribosome hibernation. Collectively, these proteome adaptations will result in metabolic inactive cells which are likely to exhibit multidrug tolerance.

Interaction of the Nitrogen Regulatory Protein GlnB (PII) with Biotin Carboxyl Carrier Protein (BCCP) Controls Acetyl-CoA Levels in the Cyanobacterium Synechocystis sp. PCC 6803.

The family of PII signal transduction proteins (members GlnB, GlnK, NifI) plays key roles in various cellular processes related to nitrogen metabolism at different functional levels. Recent studies implied that PII proteins may also be involved in the regulation of fatty acid metabolism, since GlnB proteins from Proteobacteria and from Arabidopsis thaliana were shown to interact with biotin carboxyl carrier protein (BCCP) of acetyl-CoA carboxylase (ACC). In case of Escherichia coli ACCase, this interaction reduces the kcat of acetyl-CoA carboxylation, which should have a marked impact on the acetyl-CoA metabolism. In this study we show that the PII protein of a unicellular cyanobacterium inhibits the biosynthetic activity of E. coli ACC and also interacts with cyanobacterial BCCP in an ATP and 2-oxoglutarate dependent manner. In a PII mutant strain of Synechocystis strain PCC 6803, the lacking control leads to reduced acetyl-CoA levels, slightly increased levels of fatty acids and formation of lipid bodies as well as an altered fatty acid composition.

2-Oxoglutarate levels control adenosine nucleotide binding by Herbaspirillum seropedicae PII proteins.

Nitrogen metabolism in Proteobacteria is controlled by the Ntr system, in which PII proteins play a pivotal role, controlling the activity of target proteins in response to the metabolic state of the cell. Characterization of the binding of molecular effectors to these proteins can provide information about their regulation. Here, the binding of ATP, ADP and 2-oxoglutarate (2-OG) to the Herbaspirillum seropedicae PII proteins, GlnB and GlnK, was characterized using isothermal titration calorimetry. Results show that these proteins can bind three molecules of ATP, ADP and 2-OG with homotropic negative cooperativity, and 2-OG binding stabilizes the binding of ATP. Results also show that the affinity of uridylylated forms of GlnB and GlnK for nucleotides is significantly lower than that of the nonuridylylated proteins. Furthermore, fluctuations in the intracellular concentration of 2-OG in response to nitrogen availability are shown. Results suggest that under nitrogen-limiting conditions, PII proteins tend to bind ATP and 2-OG. By contrast, after an ammonium shock, a decrease in the 2-OG concentration is observed causing a decrease in the affinity of PII proteins for ATP. This phenomenon may facilitate the exchange of ATP for ADP on the ligand-binding pocket of PII proteins, thus it is likely that under low ammonium, low 2-OG levels would favor the ADP-bound state.

The bacterial signal transduction protein GlnB regulates the committed step in fatty acid biosynthesis by acting as a dissociable regulatory subunit of acetyl-CoA carboxylase.

Biosynthesis of fatty acids is one of the most fundamental biochemical pathways in nature. In bacteria and plant chloroplasts, the committed and rate-limiting step in fatty acid biosynthesis is catalyzed by a multi-subunit form of the acetyl-CoA carboxylase enzyme (ACC). This enzyme carboxylates acetyl-CoA to produce malonyl-CoA, which in turn acts as the building block for fatty acid elongation. In Escherichia coli, ACC is comprised of three functional modules: the biotin carboxylase (BC), the biotin carboxyl carrier protein (BCCP) and the carboxyl transferase (CT). Previous data showed that both bacterial and plant BCCP interact with signal transduction proteins belonging to the PII family. Here we show that the GlnB paralogues of the PII proteins from E. coli and Azospirillum brasiliense, but not the GlnK paralogues, can specifically form a ternary complex with the BC-BCCP components of ACC. This interaction results in ACC inhibition by decreasing the enzyme turnover number. Both the BC-BCCP-GlnB interaction and ACC inhibition were relieved by 2-oxoglutarate and by GlnB uridylylation. We propose that the GlnB protein acts as a 2-oxoglutarate-sensitive dissociable regulatory subunit of ACC in Bacteria.

Search for novel targets of the PII signal transduction protein in Bacteria identifies the BCCP component of acetyl-CoA carboxylase as a PII binding partner.

The PII family comprises a group of widely distributed signal transduction proteins. The archetypal function of PII is to regulate nitrogen metabolism in bacteria. As PII can sense a range of metabolic signals, it has been suggested that the number of metabolic pathways regulated by PII may be much greater than described in the literature. In order to provide experimental evidence for this hypothesis a PII protein affinity column was used to identify PII targets in Azospirillum brasilense. One of the PII partners identified was the biotin carboxyl carrier protein (BCCP), a component of the acetyl-CoA carboxylase which catalyses the committed step in fatty acid biosynthesis. As BCCP had been previously identified as a PII target in Arabidopsis thaliana we hypothesized that the PII -BCCP interaction would be conserved throughout Bacteria. In vitro experiments using purified proteins confirmed that the PII -BCCP interaction is conserved in Escherichia coli. The BCCP-PII interaction required MgATP and was dissociated by increasing 2-oxoglutarate. The interaction was modestly affected by the post-translational uridylylation status of PII ; however, it was completely dependent on the post-translational biotinylation of BCCP.

The nitrogenase regulatory enzyme dinitrogenase reductase ADP-ribosyltransferase (DraT) is activated by direct interaction with the signal transduction protein GlnB.

Fe protein (dinitrogenase reductase) activity is reversibly inactivated by dinitrogenase reductase ADP-ribosyltransferase (DraT) in response to an increase in the ammonium concentration or a decrease in cellular energy in Azospirillum brasilense, Rhodospirillum rubrum, and Rhodobacter capsulatus. The ADP-ribosyl is removed by the dinitrogenase reductase-activating glycohydrolase (DraG), promoting Fe protein reactivation. The signaling pathway leading to DraT activation by ammonium is still not completely understood, but the available evidence shows the involvement of direct interaction between the enzyme and the nitrogen-signaling P(II) proteins. In A. brasilense, two P(II) proteins, GlnB and GlnZ, were identified. We used Fe protein from Azotobacter vinelandii as the substrate to assess the activity of A. brasilense DraT in vitro complexed or not with P(II) proteins. Under our conditions, GlnB was necessary for DraT activity in the presence of Mg-ADP. The P(II) effector 2-oxoglutarate, in the presence of Mg-ATP, inhibited DraT-GlnB activity, possibly by inducing complex dissociation. DraT was also activated by GlnZ and by both uridylylated P(II) proteins, but not by a GlnB variant carrying a partial deletion of the T loop. Kinetics studies revealed that the A. brasilense DraT-GlnB complex was at least 18-fold more efficient than DraT purified from R. rubrum, but with a similar K(m) value for NAD(+). Our results showed that ADP-ribosylation of the Fe protein does not affect the electronic state of its metal cluster and prevents association between the Fe and MoFe proteins, thus inhibiting electron transfer.

Influence of the ADP/ATP ratio, 2-oxoglutarate and divalent ions on Azospirillum brasilense PII protein signalling.

Proteins belonging to the P(II) family coordinate cellular nitrogen metabolism by direct interaction with a variety of enzymes, transcriptional regulators and transporters. The sensing function of P(II) relies on its ability to bind the nitrogen/carbon signalling molecule 2-oxoglutarate (2-OG). In Proteobacteria, P(II) is further subject to reversible uridylylation according to the intracellular levels of glutamine, which reflect the cellular nitrogen status. A number of P(II) proteins have been shown to bind ADP and ATP in a competitive manner, suggesting that P(II) might act as an energy sensor. Here, we analyse the influence of the ADP/ATP ratio, 2-OG levels and divalent metal ions on in vitro uridylylation of the Azospirillum brasilense P(II) proteins GlnB and GlnZ, and on interaction with their targets AmtB, DraG and DraT. The results support the notion that the cellular concentration of 2-OG is a key factor governing occupation of the GlnB and GlnZ nucleotide binding sites by ATP or ADP, with high 2-OG levels favouring the occupation of P(II) by ATP. Both P(II) uridylylation and interaction with target proteins responded to the ADP/ATP ratio within the expected physiological range, supporting the concept that P(II) proteins might act as cellular energy sensors.

Heat stability of Proteobacterial PII protein facilitate purification using a single chromatography step.

The P(II) proteins comprise a family of widely distributed signal transduction proteins that integrate the signals of cellular nitrogen, carbon and energy status, and then regulate, by protein-protein interaction, the activity of a variety of target proteins including enzymes, transcriptional regulators and membrane transporters. We have previously shown that the P(II) proteins from Azospirillum brasilense, GlnB and GlnZ, do not alter their migration behavior under native gel electrophoresis following incubated for a few minutes at 95°C. This data suggested that P(II) proteins were either resistant to high temperatures and/or that they could return to their native state after having been unfolded by heat. Here we used (1)H NMR to show that the A. brasilense GlnB is stable up to 70°C. The melting temperature (Tm) of GlnB was determined to be 84°C using the fluorescent dye Sypro-Orange. P(II) proteins from other Proteobacteria also showed a high Tm. We exploited the thermo stability of P(II) by introducing a thermal treatment step in the P(II) purification protocol, this step significantly improved the homogeneity of A. brasilense GlnB and GlnZ, Herbaspirillum seropedicae GlnB and GlnK, and of Escherichia coli GlnK. Only a single chromatography step was necessary to obtain homogeneities higher than 95%. NMR(1) and in vitro uridylylation analysis showed that A. brasilense GlnB purified using the thermal treatment maintained its folding and activity. The purification protocol described here can facilitate the study of P(II) protein family members.

Crystal structure of the GlnZ-DraG complex reveals a different form of PII-target interaction.

Nitrogen metabolism in bacteria and archaea is regulated by a ubiquitous class of proteins belonging to the P(II)family. P(II) proteins act as sensors of cellular nitrogen, carbon, and energy levels, and they control the activities of a wide range of target proteins by protein-protein interaction. The sensing mechanism relies on conformational changes induced by the binding of small molecules to P(II) and also by P(II) posttranslational modifications. In the diazotrophic bacterium Azospirillum brasilense, high levels of extracellular ammonium inactivate the nitrogenase regulatory enzyme DraG by relocalizing it from the cytoplasm to the cell membrane. Membrane localization of DraG occurs through the formation of a ternary complex in which the P(II) protein GlnZ interacts simultaneously with DraG and the ammonia channel AmtB. Here we describe the crystal structure of the GlnZ-DraG complex at 2.1 Å resolution, and confirm the physiological relevance of the structural data by site-directed mutagenesis. In contrast to other known P(II) complexes, the majority of contacts with the target protein do not involve the T-loop region of P(II). Hence this structure identifies a different mode of P(II) interaction with a target protein and demonstrates the potential for P(II) proteins to interact simultaneously with two different targets. A structural model of the AmtB-GlnZ-DraG ternary complex is presented. The results explain how the intracellular levels of ATP, ADP, and 2-oxoglutarate regulate the interaction between these three proteins and how DraG discriminates GlnZ from its close paralogue GlnB.