PII-like signaling proteins: a new paradigm in orchestrating cellular homeostasis.

Members of the PII superfamily are versatile, multitasking signaling proteins ubiquitously found in all domains of life. They adeptly monitor and synchronize the cell's carbon, nitrogen, energy, redox, and diurnal states, primarily by binding interdependently to adenyl-nucleotides, including charged nucleotides (ATP, ADP, and AMP) and second messengers such as cyclic adenosine monophosphate (cAMP), cyclic di-adenosine monophosphate (c-di-AMP), and S-adenosylmethionine-AMP (SAM-AMP). These proteins also undergo a variety of posttranslational modifications, such as phosphorylation, adenylation, uridylation, carboxylation, and disulfide bond formation, which further provide cues on the metabolic state of the cell. Serving as precise metabolic sensors, PII superfamily proteins transmit this information to diverse cellular targets, establishing dynamic regulatory assemblies that fine-tune cellular homeostasis. Recently discovered, PII-like proteins are emerging families of signaling proteins that, while related to canonical PII proteins, have evolved to fulfill a diverse range of cellular functions, many of which remain elusive. In this review, we focus on the evolution of PII-like proteins and summarize the molecular mechanisms governing the assembly dynamics of PII complexes, with a special emphasis on the PII-like protein SbtB.

Allosteric regulation of nitrate transporter NRT via the signaling protein PII.

Coordinated carbon and nitrogen metabolism is crucial for bacteria living in the fluctuating environments. Intracellular carbon and nitrogen homeostasis is maintained by a sophisticated network, in which the widespread signaling protein PII acts as a major regulatory hub. In cyanobacteria, PII was proposed to regulate the nitrate uptake by an ABC (ATP-binding cassette)-type nitrate transporter NrtABCD, in which the nucleotide-binding domain of NrtC is fused with a C-terminal regulatory domain (CRD). Here, we solved three cryoelectron microscopy structures of NrtBCD, bound to nitrate, ATP, and PII, respectively. Structural and biochemical analyses enable us to identify the key residues that form a hydrophobic and a hydrophilic cavity along the substrate translocation channel. The core structure of PII, but not the canonical T-loop, binds to NrtC and stabilizes the CRD, making it visible in the complex structure, narrows the substrate translocation channel in NrtB, and ultimately locks NrtBCD at an inhibited inward-facing conformation. Based on these results and previous reports, we propose a putative transport cycle driven by NrtABCD, which is allosterically inhibited by PII in response to the cellular level of 2-oxoglutarate. Our findings provide a distinct regulatory mechanism of ABC transporter via asymmetrically binding to a signaling protein.

Carbon signaling protein SbtB possesses atypical redox-regulated apyrase activity to facilitate regulation of bicarbonate transporter SbtA.

The PII superfamily consists of widespread signal transduction proteins found in all domains of life. In addition to canonical PII proteins involved in C/N sensing, structurally similar PII-like proteins evolved to fulfill diverse, yet poorly understood cellular functions. In cyanobacteria, the bicarbonate transporter SbtA is co-transcribed with the conserved PII-like protein, SbtB, to augment intracellular inorganic carbon levels for efficient CO2 fixation. We identified SbtB as a sensor of various adenine nucleotides including the second messenger nucleotides cyclic AMP (cAMP) and c-di-AMP. Moreover, many SbtB proteins possess a C-terminal extension with a disulfide bridge of potential redox-regulatory function, which we call R-loop. Here, we reveal an unusual ATP/ADP apyrase (diphosphohydrolase) activity of SbtB that is controlled by the R-loop. We followed the sequence of hydrolysis reactions from ATP over ADP to AMP in crystallographic snapshots and unravel the structural mechanism by which changes of the R-loop redox state modulate apyrase activity. We further gathered evidence that this redox state is controlled by thioredoxin, suggesting that it is generally linked to cellular metabolism, which is supported by physiological alterations in site-specific mutants of the SbtB protein. Finally, we present a refined model of how SbtB regulates SbtA activity, in which both the apyrase activity and its redox regulation play a central role. This highlights SbtB as a central switch point in cyanobacterial cell physiology, integrating not only signals from the energy state (adenyl-nucleotide binding) and the carbon supply via cAMP binding but also from the day/night status reported by the C-terminal redox switch.

PotN represents a novel energy-state sensing PII subfamily, occurring in firmicutes.

PII proteins are signal processor proteins that regulate the cellular metabolism of Bacteria, Archea and plant chloroplasts typically in response to the cellular nitrogen status. Here, we report the first biochemical characterization of a novel PII-like protein PotN from Lentilactobacillus hilgardii. PotN is encoded in an operon together with the potABCD genes, encoding the ABC transporter for spermidine/putrescine. Like canonical PII proteins, the native PotN has a trimeric structure and competitively binds ATP and ADP, but it does not bind 2-oxoglutarate. Immunoprecipitation and pull-down experiments revealed that PotN is associated in vivo with the transcriptional regulator GlnR and the beta-subunit of pyruvate/2-oxoglutarate/acetoin dehydrogenase AcoB. Moreover, in vitro assays revealed that the ATPase domain of PotA also is able to interact with PotN. Interaction analyses demonstrated that PotN preferentially associates with PotA in the ADP state, whereas it binds to GlnR at elevated ATP levels. This suggests that PotN regulates the transport of polyamines and GlnR-dependent gene expression in response to the energy availability for the cell.

Regulation of Herbaspirillum seropedicae NifA by the GlnK PII signal transduction protein is mediated by effectors binding to allosteric sites.

Herbaspirillum seropedicae is a plant growth promoting bacterium that is able to fix nitrogen and to colonize the surface and internal tissues of important crops. Nitrogen fixation in H. seropedicae is regulated at the transcriptional level by the prokaryotic enhancer binding protein NifA. The activity of NifA is negatively affected by oxygen and positively stimulated by interaction with GlnK, a PII signaling protein that monitors intracellular levels of the key metabolite 2-oxoglutarate (2-OG) and functions as an indirect sensor of the intracellular nitrogen status. GlnK is also subjected to a cycle of reversible uridylylation in response to intracellular levels of glutamine. Previous studies have established the role of the N-terminal GAF domain of NifA in intramolecular repression of NifA activity and the role of GlnK in relieving this inhibition under nitrogen-limiting conditions. However, the mechanism of this control of NifA activity is not fully understood. Here, we constructed a series of GlnK variants to elucidate the role of uridylylation and effector binding during the process of NifA activation. Our data support a model whereby GlnK uridylylation is not necessary to activate NifA. On the other hand, binding of 2-OG and MgATP to GlnK are very important for NifA activation and constitute the most important signal of cellular nitrogen status to NifA.

Energy drives the dynamic localization of cyanobacterial nitrogen regulators during diurnal cycles.

Cyanobacteria, phototrophic organisms performing oxygenic photosynthesis, must adapt their metabolic processes to the challenges imposed by the succession of days and nights. Two conserved cyanobacterial proteins, PII and PipX, function as hubs of the nitrogen interaction network, forming complexes with a variety of diverse targets. While PII proteins are found in all three domains of life as integrators of signals of the nitrogen and carbon balance, PipX proteins are unique to cyanobacteria, where they provide a mechanistic link between PII signalling and the control of gene expression by the global nitrogen regulator NtcA. Here we demonstrate that PII and PipX display distinct localization patterns during diurnal cycles, co-localizing into the same foci at the periphery and poles of the cells during dark periods, a circadian-independent process requiring a low ATP/ADP ratio. Genetic, cellular biology and biochemical approaches used here provide new insights into the nitrogen regulatory network, calling attention to the roles of PII as energy sensors and its interactions with PipX in the context of essential signalling pathways. This study expands the contribution of the nitrogen regulators PII and PipX to integrate and transduce key environmental signals that allow cyanobacteria to thrive in our planet.

PII Protein-Derived FRET Sensors for Quantification and Live-Cell Imaging of 2-Oxoglutarate.

The citric acid cycle intermediate 2-oxoglutarate (2-OG, a.k.a. alpha-ketoglutarate) links the carbon and nitrogen metabolic pathways and can provide information on the metabolic status of cells. In recent years, it has become exceedingly clear that 2-OG also acts as a master regulator of diverse biologic processes in all domains of life. Consequently, there is a great demand for time-resolved data on 2-OG fluctuations that can't be adequately addressed using established methods like mass spectrometry-based metabolomics analysis. Therefore, we set out to develop a novel intramolecular 2-OG FRET sensor based on the signal transduction protein PII from Synechococcus elongatus PCC 7942. We created two variants of the sensor, with a dynamic range for 2-OG from 0.1 µM to 0.1 mM or from 10 µM to 10 mM. As proof of concept, we applied the sensors to determine in situ glutamine:2-oxoglutarate aminotransferase (GOGAT) activity in Synechococcus elongatus PCC 7942 cells and measured 2-OG concentrations in cell extracts from Escherichia coli in vitro. Finally, we could show the sensors' functionality in living human cell lines, demonstrating their potential in the context of mechanistic studies and drug screening.

Effects of T-loop modification on the PII-signalling protein: structure of uridylylated Escherichia coli GlnB bound to ATP.

To adapt to environments with variable nitrogen sources and richness, the widely distributed homotrimeric PII signalling proteins bind their allosteric effectors ADP/ATP/2-oxoglutarate, and experience nitrogen-sensitive uridylylation of their flexible T-loops at Tyr51, regulating their interactions with effector proteins. To clarify whether uridylylation triggers a given T-loop conformation, we determined the crystal structure of the classical paradigm of PII protein, Escherichia coli GlnB (EcGlnB), in fully uridylylated form (EcGlnB-UMP3 ). This is the first structure of a postranslationally modified PII protein. This required recombinant production and purification of the uridylylating enzyme GlnD and its use for full uridylylation of large amounts of recombinantly produced pure EcGlnB. Unlike crystalline non-uridylylated EcGlnB, in which T-loops are fixed, uridylylation rendered the T-loop highly mobile because of loss of contacts mediated by Tyr51, with concomitant abolition of T-loop anchoring via Arg38 on the ATP site. This site was occupied by ATP, providing the first, long-sought snapshot of the EcGlnB-ATP complex, connecting ATP binding with T-loop changes. Inferences are made on the mechanisms of PII selectivity for ATP and of PII-UMP3 signalling, proposing a model for the architecture of the complex of EcGlnB-UMP3 with the uridylylation-sensitive PII target ATase (which adenylylates/deadenylylates glutamine synthetase [GS]) and with GS.

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.

Energy Sensing versus 2-Oxoglutarate Dependent ATPase Switch in the Control of Synechococcus PII Interaction with Its Targets NAGK and PipX.

PII proteins constitute a superfamily of highly conserved signaling devices, common in all domains of life. Through binding of the metabolites ATP, ADP and 2-oxoglutarate (2-OG), they undergo conformational changes which allow them to regulate a variety of target proteins including enzymes, transport proteins and transcription factors. But, in reverse, these target proteins also modulate the metabolite sensing properties of PII, as has been recently shown. We used this effect to refine our PII based Förster resonance energy transfer (FRET) sensor and amplify its sensitivity towards ADP. With this enhanced sensor setup we addressed the question whether the PII protein from the model organism Synechococcus elongatus autonomously switches into the ADP conformation through ATPase activity as proposed in a recently published model. The present study disproves ATPase activity as a relevant mechanism for the transition of PII into the ADP state. In the absence of 2-OG, only the ATP/ADP ratio and concentration of ADP directs the competitive interaction of PII with two targets, one of which preferentially binds PII in the ATP-state, the other in the ADP-state.

Nitrogen stress response and stringent response are coupled in Escherichia coli.

Assimilation of nitrogen is an essential process in bacteria. The nitrogen regulation stress response is an adaptive mechanism used by nitrogen-starved Escherichia coli to scavenge for alternative nitrogen sources and requires the global transcriptional regulator NtrC. In addition, nitrogen-starved E. coli cells synthesize a signal molecule, guanosine tetraphosphate (ppGpp), which serves as an effector molecule of many processes including transcription to initiate global physiological changes, collectively termed the stringent response. The regulatory mechanisms leading to elevated ppGpp levels during nutritional stresses remain elusive. Here, we show that transcription of relA, a key gene responsible for the synthesis of ppGpp, is activated by NtrC during nitrogen starvation. The results reveal that NtrC couples these two major bacterial stress responses to manage conditions of nitrogen limitation, and provide novel mechanistic insights into how a specific nutritional stress leads to elevating ppGpp levels 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.

From PII signaling to metabolite sensing: a novel 2-oxoglutarate sensor that details PII-NAGK complex formation.

The widespread PII signal transduction proteins are known for integrating signals of nitrogen and energy supply and regulating cellular behavior by interacting with a multitude of target proteins. The PII protein of the cyanobacterium Synechococcus elongatus forms complexes with the controlling enzyme of arginine synthesis, N-acetyl-L-glutamate kinase (NAGK) in a 2-oxoglutarate- and ATP/ADP-dependent manner. Fusing NAGK and PII proteins to either CFP or YFP yielded a FRET sensor that specifically responded to 2-oxoglutarate. The impact of the fluorescent tags on PII and NAGK was evaluated by enzyme assays, surface plasmon resonance spectroscopy and isothermal calorimetric experiments. The developed FRET sensor provides real-time data on PII - NAGK interaction and its modulation by the effector molecules ATP, ADP and 2-oxoglutarate in vitro. Additionally to its utility to monitor 2-oxoglutarate levels, the FRET assay provided novel insights into PII - NAGK complex formation: (i) It revealed the formation of an encounter-complex between PII and NAGK, which holds the proteins in proximity even in the presence of inhibitors of complex formation; (ii) It revealed that the PII T-loop residue Ser49 is neither essential for complex formation with NAGK nor for activation of the enzyme but necessary to form a stable complex and efficiently relieve NAGK from arginine inhibition; (iii) It showed that arginine stabilizes the NAGK hexamer and stimulates PII - NAGK interaction.

Probing the amino acids critical for protein oligomerisation and protein-nucleotide interaction in Mycobacterium tuberculosis PII protein through integration of computational and experimental approaches.

We investigated the interacting amino acids critical for the stability and ATP binding of Mycobacterium tuberculosis PII protein through a series of site specific mutagenesis experiments. We assessed the effect of mutants using glutaraldehyde crosslinking and size exclusion chromatography and isothermal titration calorimetry. Mutations in the amino acid pair R60-E62 affecting central electrostatic interaction resulted in insoluble proteins. Multiple sequence alignment of PII orthologs displayed a conserved pattern of charged residues at these positions. Mutation of amino acid D97 to a neutral residue was tolerated whereas positive charge was not acceptable. Mutation of R107 alone had no effect on trimer formation. However, the combination of neutral residues both at positions 97 and 107 was not acceptable even with the pair at 60-62 intact. Reversal of charge polarity could partially restore the interaction. The residues including K90, R101 and R103 with potential to form H-bonds to ATP are conserved throughout across numerous orthologs of PII but when mutated to Alanine, they did not show significant differences in the total free energy change of the interaction as examined through isothermal titration calorimetry. The ATP binding pattern showed anti-cooperativity using three-site binding model. We observed compensatory effect in enthalpy and entropy changes and these may represent structural adjustments to accommodate ATP in the cavity even in absence of some interactions to perform the requisite function. In this respect these small differences between the PII orthologs may have evolved to suite species specific physiological niches.

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.

Mathematical model of the binding of allosteric effectors to the Escherichia coli PII signal transduction protein GlnB.

PII proteins are important regulators of nitrogen metabolism in a wide variety of organisms: the binding of the allosteric effectors ATP, ADP, and 2-oxoglutarate (2-OG) to PII proteins affects their ability to interact with target proteins. We modeled the simultaneous binding of ATP, ADP, and 2-OG to one PII protein, namely GlnB of Escherichia coli, using a modeling approach that allows the prediction of the proportions of individual binding states. Four models with different binding rules were compared. We selected one of these models (that assumes that the binding of the first nucleotide to GlnB makes it harder for subsequent nucleotides to bind) and used it to explore how physiological concentrations of ATP, ADP, and 2-OG would affect the proportions of those states of GlnB that interact with the target proteins ATase and NtrB. Our simulations indicate that GlnB can, as suggested by previous researchers, act as a sensor of both 2-OG and the ATP:ADP ratio. We conclude that our modeling approach will be an important tool in future studies concerning the PII binding states and their interactions with target proteins.

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.

Uridylylation of Herbaspirillum seropedicae GlnB and GlnK proteins is differentially affected by ATP, ADP and 2-oxoglutarate in vitro.

PII are signal-transducing proteins that integrate metabolic signals and transmit this information to a large number of proteins. In proteobacteria, PII are modified by GlnD (uridylyltransferase/uridylyl-removing enzyme) in response to the nitrogen status. The uridylylation/deuridylylation cycle of PII is also regulated by carbon and energy signals such as ATP, ADP and 2-oxoglutarate (2-OG). These molecules bind to PII proteins and alter their tridimensional structure/conformation and activity. In this work, we determined the effects of ATP, ADP and 2-OG levels on the in vitro uridylylation of Herbaspirillum seropedicae PII proteins, GlnB and GlnK. Both proteins were uridylylated by GlnD in the presence of ATP or ADP, although the uridylylation levels were higher in the presence of ATP and under high 2-OG levels. Under excess of 2-OG, the GlnB uridylylation level was higher in the presence of ATP than with ADP, while GlnK uridylylation was similar with ATP or ADP. Moreover, in the presence of ADP/ATP molar ratios varying from 10/1 to 1/10, GlnB uridylylation level decreased as ADP concentration increased, whereas GlnK uridylylation remained constant. The results suggest that uridylylation of both GlnB and GlnK responds to 2-OG levels, but only GlnB responds effectively to variation on ADP/ATP ratio.