PII Signal Transduction Protein Research Articles

Structure of the PII signal transduction protein of Neisseria meningitidis at 1.85 A resolution.

The P(II) signal transduction proteins GlnB and GlnK are implicated in the regulation of nitrogen assimilation in Escherichia coli and other enteric bacteria. P(II)-like proteins are widely distributed in bacteria, archaea and plants. In contrast to other bacteria, Neisseria are limited to a single P(II) protein (NMB 1995), which shows a high level of sequence identity to GlnB and GlnK from Escherichia coli (73 and 62%, respectively). The structure of the P(II) protein from N. meningitidis (serotype B) has been solved by molecular replacement to a resolution of 1.85 A. Comparison of the structure with those of other P(II) proteins shows that the overall fold is tightly conserved across the whole population of related proteins, in particular the positions of the residues implicated in ATP binding. It is proposed that the Neisseria P(II) protein shares functions with GlnB/GlnK of enteric bacteria.

P II -Regulated Arginine Synthesis Controls Accumulation of Cyanophycin in Synechocystis sp. Strain PCC 6803

Cyanophycin (multi-L-arginyl-poly-L-aspartic acid) is a nitrogen storage polymer found in most cyanobacteria and some heterotrophic bacteria. The cyanobacterium Synechocystis sp. strain PCC 6803 accumulates cyanophycin following a transition from nitrogen-limited to nitrogen-excess conditions. Here we show that the accumulation of cyanophycin depends on the activation of the key enzyme of arginine biosynthesis, N-acetyl-L-glutamate kinase, by signal transduction protein PII.

The PII Signal Transduction Protein of Arabidopsis thaliana Forms an Arginine-regulated Complex with Plastid N-Acetyl Glutamate Kinase

The PII proteins are key mediators of the cellular response to carbon and nitrogen status and are found in all domains of life. In eukaryotes, PII has only been identified in red algae and plants, and in these organisms, PII localizes to the plastid. PII proteins perform their role by assessing cellular carbon, nitrogen, and energy status and conferring this information to other proteins through protein-protein interaction. We have used affinity chromatography and mass spectrometry to identify the PII-binding proteins of Arabidopsis thaliana. The major PII-interacting protein is the chloroplast-localized enzyme N-acetyl glutamate kinase, which catalyzes the key regulatory step in the pathway to arginine biosynthesis. The interaction of PII with N-acetyl glutamate kinase was confirmed through pull-down, gel filtration, and isothermal titration calorimetry experiments, and binding was shown to be enhanced in the presence of the downstream product, arginine. Enzyme kinetic analysis showed that PII increases N-acetyl glutamate kinase activity slightly, but the primary function of binding is to relieve inhibition of enzyme activity by the pathway product, arginine. Knowing the identity of PII-binding proteins across a spectrum of photosynthetic and non-photosynthetic organisms provides a framework for a more complete understanding of the function of this highly conserved signaling protein.

Ammonium assimilation in cyanobacteria

In cyanobacteria, after transport by specific permeases, ammonium is incorporated into carbon skeletons by the sequential action of glutamine synthetase (GS) and glutamate synthase (GOGAT). Two types of GS (GSI and GSIII) and two types of GOGAT (ferredoxin-GOGAT and NADH-GOGAT) have been characterized in cyanobacteria. The carbon skeleton substrate of the GS-GOGAT pathway is 2-oxoglutarate that is synthesized by the isocitrate dehydrogenase (IDH). In order to maintain the C-N balance and the amino acid pools homeostasis, ammonium assimilation is tightly regulated. The key regulatory point is the GS, which is controlled at transcriptional and posttranscriptional levels. The transcription factor NtcA plays a critical role regulating the expression of the GS and the IDH encoding genes. In the unicellular cyanobacterium Synechocystis sp. PCC 6803, NtcA controls also the expression of two small proteins (IF7 and IF17) that inhibit the activity of GS by direct protein-protein interaction. Cyanobacteria perceive nitrogen status by sensing the intracellular concentration of 2-oxoglutarate, a signaling metabolite that is able to modulate allosterically the function of NtcA, in vitro. In vivo, a functional dependence between NtcA and the signal transduction protein PII in controlling NtcA-dependent genes has been also shown.

Nitrogen assimilation and nitrogen control in cyanobacteria

Nitrogen sources commonly used by cyanobacteria include ammonium, nitrate, nitrite, urea and atmospheric N(2), and some cyanobacteria can also assimilate arginine or glutamine. ABC (ATP-binding cassette)-type permeases are involved in the uptake of nitrate/nitrite, urea and most amino acids, whereas secondary transporters take up ammonium and, in some strains, nitrate/nitrite. In cyanobacteria, nitrate and nitrite reductases are ferredoxin-dependent enzymes, arginine is catabolized by a combination of the urea cycle and arginase pathway, and urea is degraded by a Ni(2+)-dependent urease. These pathways provide ammonium that is incorporated into carbon skeletons through the glutamine synthetase-glutamate synthase cycle, in which 2-oxoglutarate is the final nitrogen acceptor. The expression of many nitrogen assimilation genes is subjected to regulation being activated by the nitrogen-control transcription factor NtcA, which is autoregulatory and whose activity appears to be influenced by 2-oxoglutarate and the signal transduction protein P(II). In some filamentous cyanobacteria, N(2) fixation takes place in specialized cells called heterocysts that differentiate from vegetative cells in a process strictly controlled by NtcA.

Complex formation between AmtB and GlnK: an ancestral role in prokaryotic nitrogen control

Ammonium transport proteins belonging to the Amt family are ubiquitous in prokaryotes. In Escherichia coli, the AmtB protein and the associated P(II) signal transduction protein (GlnK) have recently been recognized as an ammonium sensory system that effectively couples the intracellular nitrogen regulation (Ntr) system to external changes in ammonium availability. Given the almost invariant coupling of AmtB and GlnK in bacteria and archaea it seems probable that these two proteins may constitute an ancestral nitrogen-responsive system that has been coupled with a variety of unrelated nitrogen regulatory processes, which are now found in prokaryotes. The multiplicity of P(II) proteins could therefore be considered to have evolved from an ancestral GlnK-like protein and to have subsequently been adapted to control many other aspects of nitrogen metabolism.

Mutations altering the N-terminal receiver domain of NRI (NtrC) That prevent dephosphorylation by the NRII-PII complex in Escherichia coli.

The phosphorylated form of NRI is the transcriptional activator of nitrogen-regulated genes in Escherichia coli. NRI approximately P displays a slow autophosphatase activity and is rapidly dephosphorylated by the complex of the NRII and PII signal transduction proteins. Here we describe the isolation of two mutations, causing the alterations DeltaD10 and K104Q in the receiver domain of NRI, that were selected as conferring resistance to dephosphorylation by the NRII-PII complex. The mutations, which alter highly conserved residues near the D54 site of phosphorylation in the NRI receiver domain, resulted in elevated expression of nitrogen-regulated genes under nitrogen-rich conditions. The altered NRI receiver domains were phosphorylated by NRII in vitro but were defective in dephosphorylation. The DeltaD10 receiver domain retained normal autophosphatase activity but was resistant to dephosphorylation by the NRII-PII complex. The K104Q receiver domain lacked both the autophosphatase activity and the ability to be dephosphorylated by the NRII-PII complex. The properties of these altered proteins are consistent with the hypothesis that the NRII-PII complex is not a true phosphatase but rather collaborates with NRI approximately P to bring about its dephosphorylation.

Interactions between the Nitrogen Signal Transduction Protein PII and N -Acetyl Glutamate Kinase in Organisms That Perform Oxygenic Photosynthesis

Interactions between the Nitrogen Signal Transduction Protein PII and <i>N</i> -Acetyl Glutamate Kinase in Organisms That Perform Oxygenic Photosynthesis

Interactions between the nitrogen signal transduction protein PII and N-acetyl glutamate kinase in organisms that perform oxygenic photosynthesis.

PII, one of the most conserved signal transduction proteins, is believed to be a key player in the coordination of nitrogen assimilation and carbon metabolism in bacteria, archaea, and plants. However, the identity of PII receptors remains elusive, particularly in photosynthetic organisms. Here we used yeast two-hybrid approaches to identify new PII receptors and to explore the extent of conservation of PII signaling mechanisms between eubacteria and photosynthetic eukaryotes. Screening of Synechococcus sp. strain PCC 7942 libraries with PII as bait resulted in identification of N-acetyl glutamate kinase (NAGK), a key enzyme in the biosynthesis of arginine. The integrity of Ser49, a residue conserved in PII proteins from organisms that perform oxygenic photosynthesis, appears to be essential for NAGK binding. The effect of glnB mutations on NAGK activity is consistent with positive regulation of NAGK by PII. Phylogenetic and yeast two-hybrid analyses strongly suggest that there was conservation of the NAGK-PII regulatory interaction in the evolution of cyanobacteria and chloroplasts, providing insight into the function of eukaryotic PII-like proteins.

Lack of evidence for phosphorylation of Arabidopsis thaliana PII: implications for plastid carbon and nitrogen signaling

The PII signal transduction protein is regulated by covalent modification in most prokaryotic organisms. In enteric bacteria PII is uridylylated on a specific tyrosine residue in the T-loop region, while in certain cyanobacteria it is phosphorylated at the serine residue two positions away from the equivalent modified tyrosine of enteric bacteria. Covalent modification functions primarily to signal cellular nitrogen status in prokaryotes. Here we have examined the phospho-status of Arabidopsis thaliana PII under various growth conditions employing a variety of techniques, including in vivo labeling, phosphospecific antibodies, protein phosphatase treatment, mass spectrometry and protein kinase assays. All results indicate that plant PII is not regulated by phosphorylation. Edman sequencing of immunoprecipitated A. thaliana PII revealed the N-terminal sequences AQISSD and QISSDY, indicating that the mature protein is cleaved from its transit peptide in vivo at the site(s) predicted by ChloroP. Western blot analysis also demonstrated that plant PII protein expression varies little with nutrient regime.

Ammonium sensing in Escherichia coli. Role of the ammonium transporter AmtB and AmtB-GlnK complex formation.

The Amt proteins are high affinity ammonium transporters that are conserved in all domains of life. In bacteria and archaea the Amt structural genes (amtB) are invariably linked to glnK, which encodes a member of the P(II) signal transduction protein family, proteins that regulate many facets of nitrogen metabolism. We have now shown that Escherichia coli AmtB is inactivated by formation of a membrane-bound complex with GlnK. Complex formation is reversible and occurs within seconds in response to micromolar changes in the extracellular ammonium concentration. Regulation is mediated by the uridylylation/deuridylylation of GlnK in direct response to fluctuations in the intracellular glutamine pool. Furthermore under physiological conditions AmtB activity is required for GlnK deuridylylation. Hence the transporter is an integral part of the signal transduction cascade, and AmtB can be formally considered to act as an ammonium sensor. This system provides an exquisitely sensitive mechanism to control ammonium flux into the cell, and the conservation of glnK linkage to amtB suggests that this regulatory mechanism may occur throughout prokaryotes.

The Evolutionarily Conserved Trimeric Structure of CutA1 Proteins Suggests a Role in Signal Transduction

CutA1 are a protein family present in bacteria, plants, and animals, including humans. Escherichia coli CutA1 is involved in copper tolerance, whereas mammalian proteins are implicated in the anchoring of acetylcholinesterase in neuronal cell membranes. The x-ray structures of CutA1 from E. coli and rat were determined. Both proteins are trimeric in the crystals and in solution through an inter-subunit beta-sheet formation. Each subunit consists of a ferredoxin-like (beta1alpha1beta2beta3alpha2beta4) fold with an additional strand (beta5), a C-terminal helix (alpha3), and an unusual extended beta-hairpin involving strands beta2 and beta3. The bacterial CutA1 is able to bind copper(II) in vitro through His2Cys coordination in a type II water-accessible site, whereas the rat protein precipitates in the presence of copper(II). The evolutionarily conserved trimeric assembly of CutA1 is reminiscent of the architecture of PII signal transduction proteins. This similarity suggests an intriguing role of CutA1 proteins in signal transduction through allosteric communications between subunits.

Genetic and biochemical analysis of phosphatase activity of Escherichia coli NRII (NtrB) and its regulation by the PII signal transduction protein.

Mutant forms of Escherichia coli NRII (NtrB) were isolated that retained wild-type NRII kinase activity but were defective in the PII-activated phosphatase activity of NRII. Mutant strains were selected as mimicking the phenotype of a strain (strain BK) that lacks both of the related PII and GlnK signal transduction proteins and thus has no mechanism for activation of the NRII phosphatase activity. The selection and screening procedure resulted in the isolation of numerous mutants that phenotypically resembled strain BK to various extents. Mutations mapped to the glnL (ntrB) gene encoding NRII and were obtained in all three domains of NRII. Two distinct regions of the C-terminal, ATP-binding domain were identified by clusters of mutations. One cluster, including the Y302N mutation, altered a lid that sits over the ATP-binding site of NRII. The other cluster, including the S227R mutation, defined a small surface on the "back" or opposite side of this domain. The S227R and Y302N proteins were purified, along with the A129T (NRII2302) protein, which has reduced phosphatase activity due to a mutation in the central domain of NRII, and the L16R protein, which has a mutation in the N-terminal domain of NRII. The S227R, Y302N, and L16R proteins were specifically defective in the PII-activated phosphatase activity of NRII. Wild-type NRII, Y302N, A129T, and L16R proteins bound to PII, while the S227R protein was defective in binding PII. This suggests that the PII-binding site maps to the "back" of the C-terminal domain and that mutation of the ATP-lid, central domain, and N-terminal domain altered functions necessary for the phosphatase activity after PII binding.

Context-dependent functions of the PII and GlnK signal transduction proteins in Escherichia coli.

Two closely related signal transduction proteins, PII and GlnK, have distinct physiological roles in the regulation of nitrogen assimilation. Here, we examined the physiological roles of PII and GlnK when these proteins were expressed from various regulated or constitutive promoters. The results indicate that the distinct functions of PII and GlnK were correlated with the timing of expression and levels of accumulation of the two proteins. GlnK was functionally converted into PII when its expression was rendered constitutive and at the appropriate level, while PII was functionally converted into GlnK by engineering its expression from the nitrogen-regulated glnK promoter. Also, the physiological roles of both proteins were altered by engineering their expression from the nitrogen-regulated glnA promoter. We hypothesize that the use of two functionally identical PII-like proteins, which have distinct patterns of expression, may allow fine control of Ntr genes over a wide range of environmental conditions. In addition, we describe results suggesting that an additional, unknown mechanism may control the cellular level of GlnK.

Herbaspirillum seropedicae signal transduction protein PII is structurally similar to the enteric GlnK.

PII-like proteins are signal transduction proteins found in bacteria, archaea and eukaryotes. They mediate a variety of cellular responses. A second PII-like protein, called GlnK, has been found in several organisms. In the diazotroph Herbaspirillum seropedicae, PII protein is involved in sensing nitrogen levels and controlling nitrogen fixation genes. In this work, the crystal structure of the unliganded H. seropedicae PII was solved by X-ray diffraction. H. seropedicae PII has a Gly residue, Gly108 preceding Pro109 and the main-chain forms a beta turn. The glycine at position 108 allows a bend in the C-terminal main-chain, thereby modifying the surface of the cleft between monomers and potentially changing function. The structure suggests that the C-terminal region of PII proteins may be involved in specificity of function, and nonenteric diazotrophs are found to have the C-terminal consensus XGXDAX(107-112). We are also proposing binding sites for ATP and 2-oxoglutarate based on the structural alignment of PII with PII-ATP/GlnK-ATP, 5-carboxymethyl-2-hydroxymuconate isomerase and 4-oxalocrotonate tautomerase bound to the inhibitor 2-oxo-3-pentynoate.

The novel protein phosphatase PphA from Synechocystis PCC 6803 controls dephosphorylation of the signalling protein PII.

The family of PII signal transduction proteins consists of one of the most highly conserved signalling proteins in nature. The cyanobacterial PII homologue transmits signals on the nitrogen and carbon status of the cells through phosphorylation of a seryl residue. Recently, we identified a protein phosphatase 2C (PP2C) homologue from the cyanobacterium Synechocystis PCC 6803, termed PphA, to be the cellular phospho-PII (PII-P) phosphatase. In this investigation, we characterized the enzymatic properties of PphA and investigated the regulation of its catalytic activity towards PII-P. PphA dephosphorylates phosphocasein and PII-P with similar efficiency in a strictly Mg2+- or Mn2+-dependent reaction. Low-molecular-weight phosphorylated molecules are poor substrates for PphA. Its reactivity towards PII-P, but not towards phosphocasein, is inhibited by various nucleotides, suggesting that this effect is based on specific properties of the PII protein. The inhibitory effect of ATP can be strongly enhanced by the addition of 2-oxoglutarate or oxaloacetate. At low concentrations of 2-oxoglutarate, changes in the ATP levels within the physiological range affect the degree of PII-Pase inhibition, whereas at 2-oxoglutarate levels beyond 0.1 mM, inhibition is almost complete at very low ATP levels. This suggests that PII dephosphorylation is not only sensitive to 2-oxoglutarate and oxaloacetate levels, it also integrates signals from the energy charge of the cells under specific cellular conditions.

Product dependence and bifunctionality compromise the ultrasensitivity of signal transduction cascades

Covalent modification cycles are ubiquitous. Theoretical studies have suggested that they serve to increase sensitivity. However, this suggestion has not been corroborated experimentally in vivo. Here, we demonstrate that the assumptions of the theoretical studies, i.e., irreversibility and absence of product inhibition, were not trivial: when the conversion reactions are close to equilibrium or saturated by their product, "zero-order" ultrasensitivity disappears. For high sensitivities to arise, not only substrate saturation (zero-order) but also high equilibrium constants and low product saturation are required. Many covalent modification cycles are catalyzed by one bifunctional 'ambiguous' enzyme rather than by two independent proteins. This makes high substrate concentration and low product concentration for both reactions of the cycle inconsistent; such modification cycles cannot have high responses. Defining signal strength as ratios of modified (e.g., phosphorylated) over unmodified protein, signal-to-signal response sensitivity equals 1: signal strength should remain constant along a cascade of ambiguous modification cycles. We also show that the total concentration of a signalling effector protein cannot affect the signal emanating from a modification cycle catalyzed by an ambiguous enzyme if the ratio of the two forms of the effector protein is not altered. This finding may explain the experimental result that the pivotal signal transduction protein PII plus its paralogue GlnK do not control steady-state N-signal transduction in Escherichia coli. It also rationalizes the absence of strong phenotypes for many signal-transduction proteins. Emphasis on extent of modification of these proteins is perhaps more urgent than transcriptome analysis.

Role of GlnK in NifL-mediated regulation of NifA activity in Azotobacter vinelandii.

In several diazotrophic species of Proteobacteria, P(II) signal transduction proteins have been implicated in the regulation of nitrogen fixation in response to NH(4)(+) by several mechanisms. In Azotobacter vinelandii, expression of nifA, encoding the nif-specific activator, is constitutive, and thus, regulation of NifA activity by the flavoprotein NifL appears to be the primary level of nitrogen control. In vitro and genetic evidence suggests that the nitrogen response involves the P(II)-like GlnK protein and GlnD (uridylyltransferase/uridylyl-removing enzyme), which reversibly uridylylates GlnK in response to nitrogen limitation. Here, the roles of GlnK and GlnK-UMP in A. vinelandii were studied to determine whether the Nif (-) phenotype of glnD strains was due to an inability to modify GlnK, an effort previously hampered because glnK is an essential gene in this organism. A glnKY51F mutation, encoding an unuridylylatable form of the protein, was stable only in a strain in which glutamine synthetase activity is not inhibited by NH(4)(+), suggesting that GlnK-UMP is required to signal adenylyltransferase/adenylyl-removing enzyme-mediated deadenylylation. glnKY51F strains were significantly impaired for diazotrophic growth and expression of a nifH-lacZ fusion. NifL interacted with GlnK and GlnKY51F in a yeast two-hybrid system. Together, these data are consistent with those obtained from in vitro experiments (Little et al., EMBO J., 19:6041-6050, 2000) and support a model for regulation of NifA activity in which unmodified GlnK stimulates NifL inhibition and uridylylation of GlnK in response to nitrogen limitation prevents this function. This model is distinct from one proposed for the related bacterium Klebsiella pneumoniae, in which unmodified GlnK relieves NifL inhibition instead of stimulating it.

A PP2C-type phosphatase dephosphorylates the PII signaling protein in the cyanobacterium Synechocystis PCC 6803.

The family of the PII signal transduction proteins contains the most highly conserved signaling proteins in nature. The cyanobacterial PII-homologue transmits signals of the cellular nitrogen status and carbon status through phosphorylation of a seryl-residue. To identify the enzyme responsible for dephosphorylation of the phosphorylated PII protein in Synechocystis PCC 6803, prospective phosphatase encoding genes were inactivated by targeted insertion of kanamycin resistance cassettes. Disruption of ORF sll1771 generates a mutant unable to dephosphorylate PII under various experimental conditions. On the basis of conserved signature motifs, the sll1771 product (termed PphA) is a member of the protein phosphatase 2C (PP2C) superfamily, which is characterized by Mg(2+)/Mn(2+)-dependent catalytic activity. Biochemical analysis of overexpressed and purified PphA confirms its PP2C-type enzymatic properties and demonstrated its reactivity toward the phosphorylated PII protein. Thus, PphA is the first protein phosphatase in Synechocystis PCC 6803 for which the physiological substrate and function is known.

Functional dissection of the dimerization and enzymatic activities of Escherichia coli nitrogen regulator II and their regulation by the PII protein.

The dimeric two-component system transmitter protein NRII (NtrB) of Escherichia coli, product of glnL (ntrB), controls transcription of nitrogen-regulated genes by catalyzing the phosphorylation and dephosphorylation of the transcription factor NRI (NtrC). Previous studies showed that the PII signal transduction protein inhibits the kinase activity of NRII and activates its phosphatase activity. We observed that PII greatly stimulated the NRII phosphatase activity under conditions where the cleavage of ATP was prevented, indicating that the phosphatase activity did not result simply from prevention of the antagonistic NRII kinase activity by PII. Rather, PII was an activator of the phosphatase activity. To study this regulation, we examined the dimerization and enzymatic activities of NRII and various polypeptides derived from NRII, and their regulation by PII. Our results were consistent with the hypothesis that NRII consists of three domains: an N-terminal domain found only in NRII proteins and two domains formed by the conserved transmitter module of NRII, the phosphotransferase/phosphatase/dimerization (central) domain and the kinase domain. All three domains were involved in regulating the kinase and phosphatase activities of NRII. The N-terminal domain was involved in intramolecular signal transduction, and controlled access to the NRII active site for the isolated dimeric central domain added in trans. The central domain was responsible for dimerization and the phosphotransferase and phosphatase activities of NRII, but the latter activity was weak in the isolated domain and was not regulated by PII. The C-terminal kinase domain was responsible for the kinase activity. The PII protein appeared to interact with the isolated transmitter module of NRII, and not with the N-terminal domain as previously thought, since PII dramatically increased the stoichiometry of autophosphorylation of the isolated transmitter module. However, the phosphatase activity of the transmitter module of NRII was low even in the presence of PII, suggesting that the N-terminal domain was necessary for the central domain to assume the conformation necessary for potent phosphatase activity. Also, PII significantly reduced the rate of transphosphorylation of the isolated central domain by the isolated kinase domain, suggesting that PII interacts directly with the kinase domain. We hypothesize that the binding of PII to the kinase domain of NRII results in an altered conformation that is transmitted to the central and N-terminal domains; this causes the central domain to assume the conformation with potent phosphatase activity.