P(II) signal transduction proteins are ATPases whose activity is regulated by 2-oxoglutarate.

P(II) proteins are one of the most widespread families of signal transduction proteins in nature, being ubiquitous throughout bacteria, archaea, and plants. In all these organisms, P(II) proteins coordinate many facets of nitrogen metabolism by interacting with and regulating the activities of enzymes, transcription factors, and membrane transport proteins. The primary mode of signal perception by P(II) proteins derives from their ability to bind the effector molecules 2-oxoglutarate (2-OG) and ATP or ADP. The role of 2-OG as an indicator of cellular nitrogen status is well understood, but the function of ATP/ADP binding has remained unresolved. We have now shown that the Escherichia coli P(II) protein, GlnK, has an ATPase activity that is inhibited by 2-OG. Hence, when a drop in the cellular 2-OG pool signals nitrogen sufficiency, 2-OG depletion of GlnK causes bound ATP to be hydrolyzed to ADP, leading to a conformational change in the protein. We propose that the role of ATP/ADP binding in E. coli GlnK is to effect a 2-OG-dependent molecular switch that drives a conformational change in the T loops of the P(II) protein. We have further shown that two other P(II) proteins, Azospirillum brasilense GlnZ and Arabidopsis thaliana P(II), have a similar ATPase activity, and we therefore suggest that this switch mechanism is likely to be a general property of most members of the P(II) protein family.

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.

PII signal transduction proteins: pivotal players in post-translational control of nitrogenase activity.

The fixation of atmospheric nitrogen by the prokaryotic enzyme nitrogenase is an energy- expensive process and consequently it is tightly regulated at a variety of levels. In many diazotrophs this includes post-translational regulation of the enzyme's activity, which has been reported in both bacteria and archaea. The best understood response is the short-term inactivation of nitrogenase in response to a transient rise in ammonium levels in the environment. A number of proteobacteria species effect this regulation through reversible ADP-ribosylation of the enzyme, but other prokaryotes have evolved different mechanisms. Here we review current knowledge of post-translational control of nitrogenase and show that, for the response to ammonium, the P(II) signal transduction proteins act as key players.

Control of AmtB-GlnK complex formation by intracellular levels of ATP, ADP, and 2-oxoglutarate.

P(II) proteins are one of the most widespread families of signal transduction proteins in nature, being ubiquitous throughout bacteria, archaea, and plants. They play a major role in coordinating nitrogen metabolism by interacting with, and regulating the activities of, a variety of enzymes, transcription factors, and membrane transport proteins. The regulatory properties of P(II) proteins derive from their ability to bind three effectors: ATP, ADP, and 2-oxoglutarate. However, a clear model to integrate physiological changes with the consequential structural changes that mediate P(II) interaction with a target protein has so far not been developed. In this study, we analyzed the fluctuations in intracellular effector pools in Escherichia coli during association and dissociation of the P(II) protein GlnK with the ammonia channel AmtB. We determined that key features promoting AmtB-GlnK complex formation are the rapid drop in the 2-oxoglutarate pool upon ammonium influx and a simultaneous, but transient, change in the ATP/ADP ratio. We were also able to replicate AmtB-GlnK interactions in vitro using the same effector combinations that we observed in vivo. This comprehensive data set allows us to propose a model that explains the way in which interactions between GlnK and its effectors influence the conformation of GlnK and thereby regulate its interaction with AmtB.

Genome-wide analysis of the role of GlnR in Streptomyces venezuelae provides new insights into global nitrogen regulation in actinomycetes.

BACKGROUND: GlnR is an atypical response regulator found in actinomycetes that modulates the transcription of genes in response to changes in nitrogen availability. We applied a global in vivo approach to identify the GlnR regulon of Streptomyces venezuelae, which, unlike many actinomycetes, grows in a diffuse manner that is suitable for physiological studies. Conditions were defined that facilitated analysis of GlnR-dependent induction of gene expression in response to rapid nitrogen starvation. Microarray analysis identified global transcriptional differences between glnR+ and glnR mutant strains under varying nitrogen conditions. To differentiate between direct and indirect regulatory effects of GlnR, chromatin immuno-precipitation (ChIP) using antibodies specific to a FLAG-tagged GlnR protein, coupled with microarray analysis (ChIP-chip), was used to identify GlnR binding sites throughout the S. venezuelae genome. RESULTS: GlnR bound to its target sites in both transcriptionally active and apparently inactive forms. Thirty-six GlnR binding sites were identified by ChIP-chip analysis allowing derivation of a consensus GlnR-binding site for S. venezuelae. GlnR-binding regions were associated with genes involved in primary nitrogen metabolism, secondary metabolism, the synthesis of catabolic enzymes and a number of transport-related functions. CONCLUSIONS: The GlnR regulon of S. venezuelae is extensive and impacts on many facets of the organism's biology. GlnR can apparently bind to its target sites in both transcriptionally active and inactive forms.

The role of effector molecules in signal transduction by PII proteins.

PII proteins are one of the most widely distributed signal transduction proteins in Nature, being ubiquitous in bacteria, archaea and plants. They act by protein-protein interaction to control the activities of a wide range of enzymes, transcription factors and transport proteins, the great majority of which are involved in cellular nitrogen metabolism. The regulatory activities of PII proteins are mediated through their ability to bind the key effector metabolites 2-OG (2-oxoglutarate), ATP and ADP. However, the molecular basis of these regulatory effects remains unclear. Recent advances in the solution of the crystal structures of PII proteins complexed with some of their target proteins, as well as the identification of the ATP/ADP- and 2-OG-binding sites, have improved our understanding of their mode of action. In all of the complex structures solved to date, the flexible T-loops of PII facilitate interaction with the target protein. The effector molecules appear to play a key role in modulating the conformation of the T-loops and thereby regulating the interactions between PII and its targets.

Substrate binding, deprotonation, and selectivity at the periplasmic entrance of the Escherichia coli ammonia channel AmtB.

The conduction mechanism of Escherichia coli AmtB, the structurally and functionally best characterized representative of the ubiquitous Amt/Rh family, has remained controversial in several aspects. The predominant view has been that it facilitates the movement of ammonium in its uncharged form as indicated by the hydrophobic nature of a pore located in the center of each subunit of the homotrimer. Using site-directed mutagenesis and a combination of biochemical and crystallographic methods, we have investigated mechanistic questions concerning the putative periplasmic ammonium ion binding site S1 and the adjacent periplasmic "gate" formed by two highly conserved phenylalanine residues, F107 and F215. Our results challenge models that propose that NH(4)(+) deprotonation takes place at S1 before NH(3) conduction through the pore. The presence of S1 confers two critical features on AmtB, both essential for its function: ammonium scavenging efficiency at very low ammonium concentration and selectivity against water and physiologically important cations. We show that AmtB activity absolutely requires F215 but not F107 and that removal or obstruction of the phenylalanine gate produces an open but inactive channel. The phenyl ring of F215 must thus play a very specific role in promoting transfer and deprotonation of substrate from S1 to the central pore. We discuss these results with respect to three distinct mechanisms of conduction that have been considered so far. We conclude that substrate deprotonation is an essential part of the conduction mechanism, but we do not rule out net electrogenic transport.

Potentials of mean force and permeabilities for carbon dioxide, ammonia, and water flux across a Rhesus protein channel and lipid membranes.

As a member of the ubiquitous ammonium transporter/methylamine permease/Rhesus (Amt/MEP/Rh) family of membrane protein channels, the 50 kDa Rhesus channel (Rh50) has been implicated in ammonia (NH(3)) and, more recently, also in carbon dioxide (CO(2)) transport. Here we present molecular dynamics simulations of spontaneous full permeation events of ammonia and carbon dioxide across Rh50 from Nitrosomonas europaea. The simulations show that Rh50 is functional in its crystallographic conformation, without the requirement for a major conformational change or the action of a protein partner. To assess the physiological relevance of NH(3) and CO(2) permeation across Rh50, we have computed potentials of mean force (PMFs) and permeabilities for NH(3) and CO(2) flux across Rh50 and compare them to permeation through a wide range of lipid membranes, either composed of pure lipids or composed of lipids plus an increasing cholesterol content. According to the PMFs, Rh50 is expected to enhance NH(3) flux across dense membranes, such as membranes with a substantial cholesterol content. Although cholesterol reduces the intrinsic CO(2) permeability of lipid membranes, the CO(2) permeabilities of all membranes studied here are too high to allow significant Rh50-mediated CO(2) flux. The increased barrier in the PMF for water permeation across Rh50 shows that Rh50 discriminates 40-fold between water and NH(3). Thus, Rh50 channels complement aquaporins, allowing the cell to regulate water and NH(3) flux independently. The PMFs for methylamine and NH(3) are virtually identical, suggesting that methylamine provides an excellent model for NH(3) in functional experiments.

The 1.3-A resolution structure of Nitrosomonas europaea Rh50 and mechanistic implications for NH3 transport by Rhesus family proteins.

The Rhesus (Rh) proteins are a family of integral membrane proteins found throughout the animal kingdom that also occur in a number of lower eukaryotes. The significance of Rh proteins derives from their presence in the human red blood cell membrane, where they constitute the second most important group of antigens used in transfusion medicine after the ABO group. Rh proteins are related to the ammonium transport (Amt) protein family and there is considerable evidence that, like Amt proteins, they function as ammonia channels. We have now solved the structure of a rare bacterial homologue (from Nitrosomonas europaea) of human Rh50 proteins at a resolution of 1.3 A. The protein is a trimer, and analysis of its subunit interface strongly argues that all Rh proteins are likely to be homotrimers and that the human erythrocyte proteins RhAG and RhCE/D are unlikely to form heterooligomers as previously proposed. When compared with structures of bacterial Amt proteins, NeRh50 shows several distinctive features of the substrate conduction pathway that support the concept that Rh proteins have much lower ammonium affinities than Amt proteins and might potentially function bidirectionally.

Organophosphate hydrolase in Brevundimonas diminuta is targeted to the periplasmic face of the inner membrane by the twin arginine translocation pathway.

A twin arginine translocation (Tat) motif, involved in transport of folded proteins across the inner membrane, was identified in the signal peptide of the membrane-associated organophosphate hydrolase (OPH) of Brevundimonas diminuta. Expression of the precursor form of OPH carrying a C-terminal His tag in an opd-negative background and subsequent immunoblotting with anti-His antibodies showed that only the mature form of OPH associated with the membrane and that the precursor form of OPH was entirely found in the cytoplasm. When OPH was expressed without the signal peptide, most of it remained in the cytoplasm, where it was apparently correctly folded and showed activity comparable to that of the membrane-associated OPH encoded by the wild-type opd gene. Amino acid substitutions in the invariant arginine residues of the Tat signal peptide affected both the processing and localization of OPH, confirming a critical role for the Tat system in membrane targeting of OPH in B. diminuta. The localization of OPH to the periplasmic face of the inner membrane in B. diminuta was demonstrated by proteinase K treatment of spheroplasts and also by fluorescence-activated cell sorting analysis of cells expressing OPH-green fluorescent protein fusions with and without an SsrA tag that targets cytoplasmic proteins to the ClpXP protease.

Evolution and functional characterization of the RH50 gene from the ammonia-oxidizing bacterium Nitrosomonas europaea.

The family of ammonia and ammonium channel proteins comprises the Amt proteins, which are present in all three domains of life with the notable exception of vertebrates, and the homologous Rh proteins (Rh50 and Rh30) that have been described thus far only in eukaryotes. The existence of an RH50 gene in bacteria was first revealed by the genome sequencing of the ammonia-oxidizing bacterium Nitrosomonas europaea. Here we have used a phylogenetic approach to study the evolution of the N. europaea RH50 gene, and we show that this gene, probably as a component of an integron cassette, has been transferred to the N. europaea genome by horizontal gene transfer. In addition, by functionally characterizing the Rh50(Ne) protein and the corresponding knockout mutant, we determined that NeRh50 can mediate ammonium uptake. The RH50(Ne) gene may thus have replaced functionally the AMT gene, which is missing in the genome of N. europaea and may be regarded as a case of nonorthologous gene displacement.

Interactions between PII proteins and the nitrogenase regulatory enzymes DraT and DraG in Azospirillum brasilense.

In Azospirillum brasilense ADP-ribosylation of dinitrogenase reductase (NifH) occurs in response to addition of ammonium to the extracellular medium and is mediated by dinitrogenase reductase ADP-ribosyltransferase (DraT) and reversed by dinitrogenase reductase glycohydrolase (DraG). The P(II) proteins GlnB and GlnZ have been implicated in regulation of DraT and DraG by an as yet unknown mechanism. Using pull-down experiments with His-tagged versions of DraT and DraG we have now shown that DraT binds to GlnB, but only to the deuridylylated form, and that DraG binds to both the uridylylated and deuridylylated forms of GlnZ. The demonstration of these specific protein complexes, together with our recent report of the ability of deuridylylated GlnZ to be sequestered to the cell membrane by the ammonia channel protein AmtB, offers new insights into the control of NifH ADP-ribosylation.

The ammonia channel protein AmtB from Escherichia coli is a polytopic membrane protein with a cleavable signal peptide.

The Escherichia coli ammonia channel protein, AmtB, is a homotrimeric polytopic inner membrane protein in which each subunit has 11 transmembrane helices. We have shown that the structural gene amtB encodes a preprotein with a signal peptide that is cleaved off to produce a topology with the N-terminus in the periplasm and the C-terminus in the cytoplasm. Deletion of the signal peptide coding region results in significantly lower levels of AmtB accumulation in the membrane but modification of the signal peptidase cleavage site, leading to aberrant cleavage, does not prevent trimer formation and does not inactivate the protein. The presence of a signal peptide is apparently not a conserved feature of all prokaryotic Amt proteins. Comparison of predicted AmtB sequences suggests that while Amt proteins in Gram-negative organisms utilize a signal peptide, the homologous proteins in Gram-positive organisms do not.

In vivo functional characterization of the Escherichia coli ammonium channel AmtB: evidence for metabolic coupling of AmtB to glutamine synthetase.

The Escherichia coli AmtB protein is member of the ubiquitous Amt family of ammonium transporters. Using a variety of [14C]methylammonium-uptake assays in wild-type E. coli, together with amtB and glutamine synthetase (glnA) mutants, we have shown that the filtration method traditionally used to measure [14C]methylammonium uptake actually measures intracellular accumulation of methylglutamine and that the kinetic data deduced from such experiments refer to the activity of glutamine synthetase and not to AmtB. Furthermore, the marked difference between the K(m) values of glutamine synthetase calculated in vitro and those calculated in vivo from our data suggest that ammonium assimilation by glutamine synthetase is coupled to the function of AmtB. The use of a modified assay technique allows us to measure AmtB activity in vivo. In this way, we have examined the role that AmtB plays in ammonium/methylammonium transport, in the light of conflicting proposals with regard to both the mode of action of Amt proteins and their substrate, i.e. ammonia or ammonium. Our in vivo data suggest that AmtB acts as a slowly conducting channel for NH3 that is neither dependent on the membrane potential nor on ATP. Furthermore, studies on competition between ammonium and methylammonium suggest that AmtB has a binding site for NH4+ on the periplasmic face.

Mutational analysis of GlnB residues critical for NifA activation in Azospirillum brasilense.

PII proteins are signal transduction that sense cellular nitrogen status and relay this signals to other targets. Azospirillum brasilense is a nitrogen fixing bacterium, which associates with grasses and cereals promoting beneficial effects on plant growth and crop yields. A. brasilense contains two PII encoding genes, named glnB and glnZ. In this paper, glnB was mutagenised in order to identify amino acid residues involved in GlnB signaling. Two variants were obtained by random mutagenesis, GlnBL13P and GlnBV100A and a site directed mutant, GlnBY51F, was obtained. Their ability to complement nitrogenase activity of glnB mutant strains of A. brasilense were determined. The variant proteins were also overexpressed in Escherichia coli, purified and characterized biochemically. None of the GlnB variant forms was able to restore nitrogenase activity in glnB mutant strains of A. brasilense LFH3 and 7628. The purified GlnBY51F and GlnBL13P proteins could not be uridylylated by GlnD, whereas GlnBV100A was uridylylated but at only 20% of the rate for wild type GlnB. Biochemical and computational analyses suggest that residue Leu13, located in the α helix 1 of GlnB, is important to maintain GlnB trimeric structure and function. The substitution V100A led to a lower affinity for ATP binding. Together the results suggest that NifA activation requires uridylylated GlnB bound to ATP.

Post-translational modification of P II signal transduction proteins.

The PII proteins constitute one of the most widely distributed families of signal transduction proteins in nature. They are pivotal players in the control of nitrogen metabolism in bacteria and archaea, and are also found in the plastids of plants. Quite remarkably PII proteins control the activities of a diverse range of enzymes, transcription factors and membrane transport proteins, and in all known cases they achieve their regulatory effect by direct interaction with their target. PII proteins in the Proteobacteria and the Actinobacteria are subject to post-translational modification by uridylylation or adenylylation respectively, whilst in some Cyanobacteria they can be modified by phosphorylation. In all these cases the protein's modification state is influenced by the cellular nitrogen status and is thought to regulate its activity. However, in many organisms there is no evidence for modification of PII proteins and indeed the ability of these proteins to respond to the cellular nitrogen status is fundamentally independent of post-translational modification. In this review we explore the role of post-translational modification in PII proteins in the light of recent studies.

Association and dissociation of the GlnK-AmtB complex in response to cellular nitrogen status can occur in the absence of GlnK post-translational modification.

PII proteins are pivotal players in the control of nitrogen metabolism in bacteria and archaea, and are also found in the plastids of plants. PII proteins control the activities of a diverse range of enzymes, transcription factors and membrane transport proteins, and their regulatory effect is achieved by direct interaction with their target. Many, but by no means all, PII proteins are subject to post-translational modification of a residue within the T-loop of the protein. The protein's modification state is influenced by the cellular nitrogen status and in the past this has been considered to regulate PII activity by controlling interaction with target proteins. However, the fundamental ability of PII proteins to respond to the cellular nitrogen status has been shown to be dependent on binding of key effector molecules, ATP, ADP, and 2-oxoglutarate which brings into question the precise role of post-translational modification. In this study we have used the Escherichia coli PII protein GlnK to examine the influence of post-translational modification (uridylylation) on the interaction between GlnK and its cognate target the ammonia channel protein AmtB. We have compared the interaction with AmtB of wild-type GlnK and a variant protein, GlnKTyr51Ala, that cannot be uridylylated. This analysis was carried out both in vivo and in vitro and showed that association and dissociation of the GlnK-AmtB complex is not dependent on the uridylylation state of GlnK. However, our in vivo studies show that post-translational modification of GlnK does influence the dynamics of its interaction with AmtB.

P(II) signal transduction proteins: nitrogen regulation and beyond.

The P(II) proteins are one of the most widely distributed families of signal transduction proteins in nature. They are pivotal players in the control of nitrogen metabolism in bacteria and archaea, and are also found in the plastids of plants. Quite remarkably, P(II) proteins control the activities of a diverse range of enzymes, transcription factors and membrane transport proteins, and in recent years the extent of these interactions has been recognized to be much greater than heretofore described. Major advances have been made in structural studies of P(II) proteins, including the solution of the first structures of P(II) proteins complexed with their targets. We have also begun to gain insights into how the key effector molecules, 2-oxoglutarate and ATP/ADP, influence the activities of P(II) proteins. In this review, we have set out to summarize our current understanding of P(II) biology and to consider where future studies of these extraordinarily adaptable proteins might lead us.

Ammonium transport proteins with changes in one of the conserved pore histidines have different performance in ammonia and methylamine conduction.

Two conserved histidine residues are located near the mid-point of the conduction channel of ammonium transport proteins. The role of these histidines in ammonia and methylamine transport was evaluated by using a combination of in vivo studies, molecular dynamics (MD) simulation, and potential of mean force (PMF) calculations. Our in vivo results showed that a single change of either of the conserved histidines to alanine leads to the failure to transport methylamine but still facilitates good growth on ammonia, whereas double histidine variants completely lose their ability to transport both methylamine and ammonia. Molecular dynamics simulations indicated the molecular basis of the in vivo observations. They clearly showed that a single histidine variant (H168A or H318A) of AmtB confines the rather hydrophobic methylamine more strongly than ammonia around the mutated sites, resulting in dysfunction in conducting the former but not the latter molecule. PMF calculations further revealed that the single histidine variants form a potential energy well of up to 6 kcal/mol for methylamine, impairing conduction of this substrate. Unlike the single histidine variants, the double histidine variant, H168A/H318A, of AmtB was found to lose its unidirectional property of transporting both ammonia and methylamine. This could be attributed to a greatly increased frequency of opening of the entrance gate formed by F215 and F107, in this variant compared to wild-type, with a resultant lowering of the energy barrier for substrate to return to the periplasm.

A new P(II) protein structure identifies the 2-oxoglutarate binding site.

P(II) proteins of bacteria, archaea, and plants regulate many facets of nitrogen metabolism. They do so by interacting with their target proteins, which can be enzymes, transcription factors, or membrane proteins. A key feature of the ability of P(II) proteins to sense cellular nitrogen status and to interact accordingly with their targets is their binding of the key metabolic intermediate 2-oxoglutarate (2-OG). However, the binding site of this ligand within P(II) proteins has been controversial. We have now solved the X-ray structure, at 1.4 A resolution, of the Azospirillum brasilense P(II) protein GlnZ complexed with MgATP and 2-OG. This structure is in excellent agreement with previous biochemical data on 2-OG binding to a variety of P(II) proteins and shows that 2-oxoglutarate binds within the cleft formed between neighboring subunits of the homotrimer. The 2-oxo acid moiety of bound 2-OG ligates the bound Mg(2+) together with three phosphate oxygens of ATP and the side chain of the T-loop residue Gln39. Our structure is in stark contrast to an earlier structure of the Methanococcus jannaschii GlnK1 protein in which the authors reported 2-OG binding to the T-loop of that P(II) protein. In the light of our new structure, three families of T-loop conformations, each associated with a distinct effector binding mode and characterized by a different interaction partner of the ammonium group of the conserved residue Lys58, emerge as a common structural basis for effector signal output by P(II) proteins.