New views on PII signaling: from nitrogen sensing to global metabolic control

PII proteins are multitasking, information-processing proteins found in all domains of life that decode the metabolic state of the cells and transduce this information to various regulatory targets.PII proteins are major regulatory hubs of cellular metabolism and orchestrate key steps of nitrogen and carbon metabolism to ensure a balanced flow of metabolites for cell growth.A range of different transporters have been recently identified as new PII targets, along with enzymes of primary metabolism, as well as enzymes required for oxaloacetate and NAD-cofactor synthesis and the synthesis of the signaling molecule (c-di-GMP).A novel class of PII-interactors occurs in cyanobacteria and archaea: small proteins without enzymatic activity that modulate cellular processes through a network of protein interactions to extend further the regulatory range of PII proteins.Understanding the mechanism of PII signal perception and transduction has allowed the development of novel in vivo metabolite-status sensors.Targeted mutations in the PII-signaling network can be used in synthetic biology to direct metabolic fluxes in desired directions for improved metabolic engineering. PII proteins are multitasking information-processing proteins occurring in bacteria, archaea, and plastids, decoding the metabolic state of the cells and providing this information to various regulatory targets. Research in recent years identified a wide range of novel PII targets mainly through ligand fishing assays, indicating that PII proteins evolved into major regulatory hubs of cellular metabolism. PII proteins orchestrate not only key steps of nitrogen and carbon metabolism but rather control a wide range of transporters and can also regulate the production of signaling molecules (c-di-GMP) and cofactors (NAD+). A recently identified class of PII-interacting proteins, which by themselves have no enzymatic activity, modulate cellular processes through protein interactions, further extending the regulatory range of PII proteins. PII proteins are multitasking information-processing proteins occurring in bacteria, archaea, and plastids, decoding the metabolic state of the cells and providing this information to various regulatory targets. Research in recent years identified a wide range of novel PII targets mainly through ligand fishing assays, indicating that PII proteins evolved into major regulatory hubs of cellular metabolism. PII proteins orchestrate not only key steps of nitrogen and carbon metabolism but rather control a wide range of transporters and can also regulate the production of signaling molecules (c-di-GMP) and cofactors (NAD+). A recently identified class of PII-interacting proteins, which by themselves have no enzymatic activity, modulate cellular processes through protein interactions, further extending the regulatory range of PII proteins. A brief retrospect on PII research – from nitrogen signaling to global metabolic controlThe first PII signaling protein was identified in the late 1960s as a small regulatory protein that affected covalent modification of glutamine synthetase activity in Escherichia coli (for comprehensive reviews on early PII research see [1.Arcondéguy T. et al.P(II) signal transduction proteins, pivotal players in microbial nitrogen control.Microbiol. Mol. Biol. Rev. 2001; 65: 80-105Crossref PubMed Scopus (348) Google Scholar,2.Huergo L.F. et al.P(II) signal transduction proteins: nitrogen regulation and beyond.FEMS Microbiol. Rev. 2013; 37: 251-283Crossref PubMed Scopus (136) Google Scholar]). For a long time, this protein was regarded as a specialized signal transduction module in Proteobacteria that responds to the carbon/nitrogen balance by sensing cellular glutamine [2.Huergo L.F. et al.P(II) signal transduction proteins: nitrogen regulation and beyond.FEMS Microbiol. Rev. 2013; 37: 251-283Crossref PubMed Scopus (136) Google Scholar] and 2-oxoglutarate (2-OG) levels [3.Fokina O. et al.Mechanism of 2-oxoglutarate signaling by the Synechococcus elongatus PII signal transduction protein.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 19760-19765Crossref PubMed Scopus (98) Google Scholar,4.Truan D. et al.A new P(II) protein structure identifies the 2-oxoglutarate binding site.J. Mol. Biol. 2010; 400: 531-539Crossref PubMed Scopus (66) Google Scholar]. More than 20 years after the first description of PII, it became obvious that PII homologues are widespread in the bacterial domain and are also present in some Archaea and in eukaryotic phototrophs [5.Leigh J.A. Dodsworth J.A. Nitrogen regulation in bacteria and archaea.Annu. Rev. Microbiol. 2007; 61: 349-377Crossref PubMed Scopus (266) Google Scholar,6.Selim K.A. et al.From cyanobacteria to Archaeplastida: new evolutionary insights into PII signalling in the plant kingdom.New Phytol. 2020; 227: 722-731Crossref PubMed Scopus (18) Google Scholar]. Furthermore, it was realized that multiple paralogues of PII may be present in prokaryotes (reviewed in [2.Huergo L.F. et al.P(II) signal transduction proteins: nitrogen regulation and beyond.FEMS Microbiol. Rev. 2013; 37: 251-283Crossref PubMed Scopus (136) Google Scholar]). In addition to the pioneering research in E. coli (reviewed in [1.Arcondéguy T. et al.P(II) signal transduction proteins, pivotal players in microbial nitrogen control.Microbiol. Mol. Biol. Rev. 2001; 65: 80-105Crossref PubMed Scopus (348) Google Scholar,2.Huergo L.F. et al.P(II) signal transduction proteins: nitrogen regulation and beyond.FEMS Microbiol. Rev. 2013; 37: 251-283Crossref PubMed Scopus (136) Google Scholar]), PII homologues were extensively studied in nitrogen-fixing bacteria [7.Dixon R. Kahn D. Genetic regulation of biological nitrogen fixation.Nat. Rev. Microbiol. 2004; 2: 621-631Crossref PubMed Scopus (702) Google Scholar] and cyanobacteria [8.Forchhammer K. Selim K.A. Carbon/nitrogen homeostasis control in cyanobacteria.FEMS Microbiol. Rev. 2020; 44: 33-53Crossref PubMed Scopus (69) Google Scholar]. By the middle of the first decade of the new millennium, PII proteins were recognized to control various reactions in anabolic nitrogen metabolism, from ammonia uptake and nitrogen fixation to arginine biosynthesis, as well as controlling nitrogen-dependent gene expression. Consequently, they were termed 'sensors of 2-OG that regulate nitrogen metabolism' [9.Ninfa A.J. Jiang P. PII signal transduction proteins: sensors of alpha-ketoglutarate that regulate nitrogen metabolism.Curr. Opin. Microbiol. 2005; 8: 168-173Crossref PubMed Scopus (204) Google Scholar]. Phylogenetic analyses [5.Leigh J.A. Dodsworth J.A. Nitrogen regulation in bacteria and archaea.Annu. Rev. Microbiol. 2007; 61: 349-377Crossref PubMed Scopus (266) Google Scholar,10.Sant'Anna F.H. et al.The PII superfamily revised: a novel group and evolutionary insights.J. Mol. Evol. 2009; 68: 322-336Crossref PubMed Scopus (63) Google Scholar] broadened our view of PII proteins by revealing that PII homologues comprise one of the most widely distributed families of signal transduction proteins in nature. A guide through the complex nomenclature of the various PII-encoding genes (glnB, glnK, glnZ, nifI) is provided in [2.Huergo L.F. et al.P(II) signal transduction proteins: nitrogen regulation and beyond.FEMS Microbiol. Rev. 2013; 37: 251-283Crossref PubMed Scopus (136) Google Scholar]. Remarkably, a new PII-encoding gene was shown to be present within an operon encoding an ABC transporter for polyamines (potABCD operon) in Lactobacillus, which was termed potN [11.Zhuravleva D.E. et al.Complete genome sequence of Lactobacillus hilgardii LMG 7934, carrying the gene encoding for the novel PII-like protein PotN.Curr. Microbiol. 2020; 77: 3538-3545Crossref PubMed Scopus (6) Google Scholar]. With the advances in protein 3D structure determination, it became evident that the family of PII signaling proteins had to be enlarged to include PII-paralogous proteins that share strong structural homology with canonical PII proteins, albeit with very limited amino acid sequence conservation [12.Forchhammer K. Lüddecke J. Sensory properties of the PII signalling protein family.FEBS J. 2016; 283: 425-437Crossref PubMed Scopus (72) Google Scholar]. These novel members of the PII superfamily with low sequence similarity to the originally described PII proteins are referred to as PII-like proteins (Box 1). The present review focuses on PII proteins that show high sequence similarity with the original PII from E. coli, and which are referred to as 'canonical PII' proteins.Box 1An expanding PII-like superfamilyBioinformatics and structural genomics approaches have greatly expanded the members of the PII superfamily, and it is proposed that the PII-like proteins represent an even more widespread family of trimeric regulators with diverse functions that occur in almost all living organisms [10.Sant'Anna F.H. et al.The PII superfamily revised: a novel group and evolutionary insights.J. Mol. Evol. 2009; 68: 322-336Crossref PubMed Scopus (63) Google Scholar,12.Forchhammer K. Lüddecke J. Sensory properties of the PII signalling protein family.FEBS J. 2016; 283: 425-437Crossref PubMed Scopus (72) Google Scholar]. We define PII-like proteins as those proteins structurally similar to canonical PII proteins with a ferredoxin-like fold but sharing low amino acid sequence conservation, including the absence of PROSITE signature sequences for canonical PII [10.Sant'Anna F.H. et al.The PII superfamily revised: a novel group and evolutionary insights.J. Mol. Evol. 2009; 68: 322-336Crossref PubMed Scopus (63) Google Scholar,12.Forchhammer K. Lüddecke J. Sensory properties of the PII signalling protein family.FEBS J. 2016; 283: 425-437Crossref PubMed Scopus (72) Google Scholar]. Several PII-like proteins have been recently identified (Figure I) and we briefly describe them in this section.The carboxysome-associated PII-like protein, which was named CPII (PDB: 5DS7), was recently characterized from the proteobacterium Thiomonas intermedia. CPII was found to bind ADP/AMP and bicarbonate and it was proposed to control carbon metabolism via sensing bicarbonate availability [74.Wheatley N.M. et al.A PII-like protein regulated by bicarbonate: structural and biochemical studies of the carboxysome-associated CPII protein.J. Mol. Biol. 2016; 428: 4013-4030Crossref PubMed Scopus (8) Google Scholar]. It is intriguing to note that, in cyanobacteria, another PII-like signaling protein, named SbtB, was also shown to control central carbon metabolism by regulating the bicarbonate transporter SbtA and the glycogen-branching enzyme GlgB [75.Selim K.A. et al.PII-like signaling protein SbtB links cAMP sensing with cyanobacterial inorganic carbon response.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E4861-E4869Crossref PubMed Scopus (33) Google Scholar, 76.Fang S. et al.Molecular mechanism underlying transport and allosteric inhibition of bicarbonate transporter SbtA.Proc. Natl. Acad. Sci. U. S. A. 2021; 118e2101632118Crossref Scopus (12) Google Scholar, 77.Selim K.A. et al.Diurnal metabolic control in cyanobacteria requires perception of second messenger signaling molecule c-di-AMP by the carbon control protein SbtB.Sci. Adv. 2021; 7eabk0568Crossref PubMed Scopus (5) Google Scholar]. SbtB proteins can bind several adenine nucleotides, including ATP, ADP, AMP, and the second-messenger nucleotides cAMP and c-di-AMP [75.Selim K.A. et al.PII-like signaling protein SbtB links cAMP sensing with cyanobacterial inorganic carbon response.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E4861-E4869Crossref PubMed Scopus (33) Google Scholar, 76.Fang S. et al.Molecular mechanism underlying transport and allosteric inhibition of bicarbonate transporter SbtA.Proc. Natl. Acad. Sci. U. S. A. 2021; 118e2101632118Crossref Scopus (12) Google Scholar, 77.Selim K.A. et al.Diurnal metabolic control in cyanobacteria requires perception of second messenger signaling molecule c-di-AMP by the carbon control protein SbtB.Sci. Adv. 2021; 7eabk0568Crossref PubMed Scopus (5) Google Scholar, 78.Kaczmarski J.A. et al.Structural basis for the allosteric regulation of the SbtA bicarbonate transporter by the PII-like protein, SbtB, from Cyanobium sp. PCC7001.Biochemistry. 2019; 58: 5030-5039Crossref PubMed Scopus (14) Google Scholar]. Likewise, other PII-like proteins, termed either PstA or DarA (PDB: 4RLE), were identified in firmicutes to bind only c-di-AMP; however, the precise functions of these proteins remain elusive [79.Gundlach J. et al.Identification, characterization, and structure analysis of the cyclic di-AMP-binding PII-like signal transduction protein DarA.J. Biol. Chem. 2015; 290: 3069-3080Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 80.Campeotto I. et al.Complex structure and biochemical characterization of the Staphylococcus aureus cyclic diadenylate monophosphate (c-di-AMP)-binding protein PstA, the founding member of a new signal transduction protein family.J. Biol. Chem. 2015; 290: 2888-2901Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 81.Müller M. et al.c-di-AMP recognition by Staphylococcus aureus PstA.FEBS Lett. 2015; 589: 45-51Crossref PubMed Scopus (42) Google Scholar, 82.Choi P.H. et al.Molecular basis for the recognition of cyclic-di-AMP by PstA, a PII-like signal transduction protein.Microbiologyopen. 2015; 4: 361-374Crossref PubMed Scopus (33) Google Scholar]. Quite strikingly, the first PII-like protein with enzymatic activity has been identified in a bacteriophage and shown to catalyze lysis of S-adenosylmethionine (SAM). The catalytic site of this PII-like SAMase is located in the lateral clefts between the subunits, resembling the nucleotide-binding pockets of canonical PII [83.Guo X. et al.Structure and mechanism of a phage-encoded SAM lyase revises catalytic function of enzyme family.eLife. 2021; 10e61818Crossref Scopus (3) Google Scholar]. The ability of various PII-like proteins to bind/hydrolyze adenine-based nucleotides concurs with the hypothesis that the primordial function of ancestral proteins of the PII superfamily was likely the interaction with adenine nucleotides, which was later extended in (most) canonical PII proteins by additional interaction of 2-OG with Mg2+-ATP-ligated PII [6.Selim K.A. et al.From cyanobacteria to Archaeplastida: new evolutionary insights into PII signalling in the plant kingdom.New Phytol. 2020; 227: 722-731Crossref PubMed Scopus (18) Google Scholar,8.Forchhammer K. Selim K.A. Carbon/nitrogen homeostasis control in cyanobacteria.FEMS Microbiol. Rev. 2020; 44: 33-53Crossref PubMed Scopus (69) Google Scholar].First indications that canonical PII proteins not only regulate nitrogen assimilatory reactions in various ways but are involved in global regulation of central metabolism came from the identification of acetyl-CoA carboxylase (ACCase, a key enzyme in fatty acid biosynthesis) as a target of PII regulation in the model plant Arabidopsis thaliana [13.Feria-Bourrellier A.B. et al.Chloroplast acetyl-CoA carboxylase activity is 2-oxoglutarate-regulated by interaction of PII with the biotin carboxyl carrier subunit.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 502-507Crossref PubMed Scopus (106) Google Scholar]. Initially regarded as a specific feature of plant PII signaling, it soon became clear that ACCase is also the target of PII regulation in many bacteria [14.Rodrigues T.E. et al.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.Mol. Microbiol. 2014; 91: 751-761Crossref PubMed Scopus (28) Google Scholar, 15.Gerhardt E.C.M. et al.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.Mol. Microbiol. 2015; 95: 1025-1035Crossref PubMed Scopus (47) Google Scholar, 16.Hauf W. et al.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.Front. Microbiol. 2016; 7: 1700Crossref PubMed Scopus (34) Google Scholar].In recent years, the range of described PII-target proteins has expanded significantly and revealed an unprecedented ability of PII proteins to interact with all kinds of cellular targets. From our current understanding of PII signaling systems, we propose that PII signaling arose early in evolution to control a set of highly conserved proteins that play central roles in anabolic reactions and thus have a wide distribution across all domains of life. Highly conserved PII interactors include the ammonium transport channel (Amt), which has been described as a PII target in a range of bacteria [17.Conroy M.J. et al.The crystal structure of the Escherichia coli AmtB-GlnK complex reveals how GlnK regulates the ammonia channel.Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 1213-1218Crossref PubMed Scopus (145) Google Scholar, 18.Gruswitz F. et al.Inhibitory complex of the transmembrane ammonia channel, AmtB, and the cytosolic regulatory protein, GlnK, at 1.96 Å.Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 42-47Crossref PubMed Scopus (104) Google Scholar, 19.Watzer B. et al.The signal transduction protein PII controls ammonium, nitrate and urea uptake in cyanobacteria.Front. Microbiol. 2019; 10: 1428Crossref PubMed Scopus (40) Google Scholar] and archaea [20.Müller M.-C. Wagner T. The oxoglutarate binding site and regulatory mechanism are conserved in ammonium transporter inhibitors GlnKs from Methanococcales.Int. J. Mol. Sci. 2021; 22: 8631Crossref PubMed Scopus (1) Google Scholar], organisms that diverged about 4 billion years ago, and the PII–ACCase complex, which was detected in plants and a range of bacteria, organisms that diverged more than 3 billion years ago [6.Selim K.A. et al.From cyanobacteria to Archaeplastida: new evolutionary insights into PII signalling in the plant kingdom.New Phytol. 2020; 227: 722-731Crossref PubMed Scopus (18) Google Scholar,13.Feria-Bourrellier A.B. et al.Chloroplast acetyl-CoA carboxylase activity is 2-oxoglutarate-regulated by interaction of PII with the biotin carboxyl carrier subunit.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 502-507Crossref PubMed Scopus (106) Google Scholar, 14.Rodrigues T.E. et al.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.Mol. Microbiol. 2014; 91: 751-761Crossref PubMed Scopus (28) Google Scholar, 15.Gerhardt E.C.M. et al.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.Mol. Microbiol. 2015; 95: 1025-1035Crossref PubMed Scopus (47) Google Scholar, 16.Hauf W. et al.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.Front. Microbiol. 2016; 7: 1700Crossref PubMed Scopus (34) Google Scholar]. Interaction of PII with N-acetyl-L-glutamate kinase (NAGK), the controlling enzyme of arginine synthesis, had long been regarded to be limited to oxygenic phototrophs (cyanobacteria and plants [6.Selim K.A. et al.From cyanobacteria to Archaeplastida: new evolutionary insights into PII signalling in the plant kingdom.New Phytol. 2020; 227: 722-731Crossref PubMed Scopus (18) Google Scholar,8.Forchhammer K. Selim K.A. Carbon/nitrogen homeostasis control in cyanobacteria.FEMS Microbiol. Rev. 2020; 44: 33-53Crossref PubMed Scopus (69) Google Scholar,21.Selim K.A. et al.Tuning the in vitro sensing and signaling properties of cyanobacterial PII protein by mutation of key residues.Sci. Rep. 2019; 9: 18985Crossref PubMed Scopus (4) Google Scholar, 22.Lapina T. et al.The PII signaling protein from red algae represents an evolutionary link between cyanobacterial and Chloroplastida PII proteins.Sci. Rep. 2018; 8: 790Crossref PubMed Scopus (15) Google Scholar, 23.Selim K.A. et al.Interaction of N-acetyl-l-glutamate kinase with the PII signal transducer in the non-photosynthetic alga Polytomella parva: Co-evolution towards a hetero-oligomeric enzyme.FEBS J. 2020; 287: 465-482Crossref PubMed Scopus (13) Google Scholar]), but recently the PII–NAGK interaction was detected in different bacterial species [24.Xu M. et al.PII signal transduction protein GlnK alleviates feedback inhibition of N-acetyl-l-glutamate kinase by l-arginine in Corynebacterium glutamicum.Appl. Environ. Microbiol. 2020; 86e00039-20Crossref Scopus (7) Google Scholar,25.Gerhardt E.C.M. et al.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.mSystems. 2020; 5e00817-20Crossref PubMed Scopus (7) Google Scholar]. Even though we cannot discard the hypothesis of more recent lateral gene transfer events, it is more likely that these PII-target protein complexes appeared in deeply branching common ancestors and were maintained during evolution. We propose that PII proteins coevolved with the majority of metabolic pathways we know today and that, during the adaptation to different lifestyles, the signaling properties of PII proteins were also adapted for specialized functions, such as nitrogen fixation in certain phylogenetic lineages. Thus, by capitalizing on their extraordinary sensory properties, PII proteins were transformed into major metabolic regulatory hubs in various bacterial lineages.The initially discovered classical PII function as regulator of glutamine synthetase through interacting with the NtrB–NtrC two-component system and the glutamine synthetase-modifying enzyme GlnE, as defined in Proteobacteria (reviewed in [1.Arcondéguy T. et al.P(II) signal transduction proteins, pivotal players in microbial nitrogen control.Microbiol. Mol. Biol. Rev. 2001; 65: 80-105Crossref PubMed Scopus (348) Google Scholar,2.Huergo L.F. et al.P(II) signal transduction proteins: nitrogen regulation and beyond.FEMS Microbiol. Rev. 2013; 37: 251-283Crossref PubMed Scopus (136) Google Scholar,5.Leigh J.A. Dodsworth J.A. Nitrogen regulation in bacteria and archaea.Annu. Rev. Microbiol. 2007; 61: 349-377Crossref PubMed Scopus (266) Google Scholar,9.Ninfa A.J. Jiang P. PII signal transduction proteins: sensors of alpha-ketoglutarate that regulate nitrogen metabolism.Curr. Opin. Microbiol. 2005; 8: 168-173Crossref PubMed Scopus (204) Google Scholar]), belongs to the specialized functions of PII proteins, as this function is absent in other phylogenetic groups. In view of the pervasive roles of PII proteins, the designation of PIIs as regulators of nitrogen metabolism is no longer a valid label for these amazingly versatile proteins (see e.g., [21.Selim K.A. et al.Tuning the in vitro sensing and signaling properties of cyanobacterial PII protein by mutation of key residues.Sci. Rep. 2019; 9: 18985Crossref PubMed Scopus (4) Google Scholar,26.Fokina O. et al.A novel signal transduction protein P(II) variant from Synechococcus elongatus PCC 7942 indicates a two-step process for NAGK-P(II) complex formation.J. Mol. Biol. 2010; 399: 410-421Crossref PubMed Scopus (36) Google Scholar, 27.Zeth K. et al.An engineered PII protein variant that senses a novel ligand: atomic resolution structure of the complex with citrate.Acta Crystallogr. D Biol. Crystallogr. 2012; 68: 901-908Crossref PubMed Scopus (17) Google Scholar, 28.Watzer B. et al.Metabolic pathway engineering using the central signal processor PII.Microb. Cell Factories. 2015; 14: 192Crossref PubMed Scopus (35) Google Scholar] for versatility of PII signaling). The time has come to provide an updated view of PII proteins as coordinators of central metabolism in prokaryotes. First, we briefly reflect on the core signaling functions of PII proteins before describing newly identified conserved functions in central metabolism. Finally, we summarize recent discoveries on specialized PII functions, as found in various phylogenetic lineages (see also Outstanding questions).The core function of PII proteins: binding of adenine nucleotidesAs described in previous reviews [6.Selim K.A. et al.From cyanobacteria to Archaeplastida: new evolutionary insights into PII signalling in the plant kingdom.New Phytol. 2020; 227: 722-731Crossref PubMed Scopus (18) Google Scholar,8.Forchhammer K. Selim K.A. Carbon/nitrogen homeostasis control in cyanobacteria.FEMS Microbiol. Rev. 2020; 44: 33-53Crossref PubMed Scopus (69) Google Scholar,12.Forchhammer K. Lüddecke J. Sensory properties of the PII signalling protein family.FEBS J. 2016; 283: 425-437Crossref PubMed Scopus (72) Google Scholar], the function of PII proteins is the sensing of the status of reporter metabolites and transducing these signals towards various PII-target proteins. This occurs through protein–protein interactions in which the PII targets perceive the conformational change in PII structure imposed by binding of the reporter metabolites. An extended, flexible loop structure (termed the T-loop) that protrudes from each subunit of the trimeric PII protein, and whose structure depends on the binding of effector molecules, plays a key role in these target interactions. The basic mode hereby is the noncovalent binding of the reporter metabolites to the 'active sites' of PII proteins. These are intercommunicating binding pockets, in the clefts between the subunits, which contact the basal part of the T-loops and have been described in detail [6.Selim K.A. et al.From cyanobacteria to Archaeplastida: new evolutionary insights into PII signalling in the plant kingdom.New Phytol. 2020; 227: 722-731Crossref PubMed Scopus (18) Google Scholar,8.Forchhammer K. Selim K.A. Carbon/nitrogen homeostasis control in cyanobacteria.FEMS Microbiol. Rev. 2020; 44: 33-53Crossref PubMed Scopus (69) Google Scholar,12.Forchhammer K. Lüddecke J. Sensory properties of the PII signalling protein family.FEBS J. 2016; 283: 425-437Crossref PubMed Scopus (72) Google Scholar]. The universally conserved trimeric architecture appears to be fundamental to the sensory properties of PII, as in this arrangement all three binding sites are directly linked with each other. A brief depiction of the sensory properties by PII proteins is provided in Figure 1.Figure 1Structure architecture and sensing properties of canonical PII proteins.Show full caption(A) Top and side view and the overall trimeric architecture of canonical PII complexed with Mg2+-ATP-2-OG (PDB:2XUL; in ribbon representation) in the metabolite-binding sites (dotted black ovals) which are located in the inter-subunit clefts. PII monomers are individually colored and the characteristic structural motifs (B- and T-loops) are indicated. Note that the large flexible T-loop is disordered and therefore not fully represented. Over the course of evolution, plant PII proteins (PDB:4USJ) acquired a C-terminal extension that forms the Gln binding site (Q-loop; shown in red), recently reviewed in [6.Selim K.A. et al.From cyanobacteria to Archaeplastida: new evolutionary insights into PII signalling in the plant kingdom.New Phytol. 2020; 227: 722-731Crossref PubMed Scopus (18) Google Scholar]. Inset. Close-up of Mg2+-ATP-2-OG binding site with relevant residues for binding shown as sticks, and H-bonds indicated by black lines. The binding of 2-OG requires previous binding of Mg2+-ATP to organize the ligation sphere for 2-OG. Hence, the ability to sense 2-OG levels likely emerged later during evolution as a secondary function of canonical PII proteins. (B) Different T-loop conformations, showing the high flexibility of the T-loop. The T-loop is the major target-binding element of canonical PII proteins and can be subject at the tip to covalent modification (phosphorylation, adenylylation, or uridylylation). The T-loop of Bacillus subtilis GlnK with ATP (yellow; PDB:4RX6), ligand-free Synechococcus elongatus GlnB (brown; PDB:1QY7), Methanocaldococcus jannaschii GlnK with ADP (blue; PDB:2J9D), Anabaena GlnB complexed with PipX and ADP (pink; PDB:5N5B), Escherichia coli GlnK ADP complexed with AmtB ammonium transporter (green; PDB:2NS1), adenylylated Corynebacterium glutamicum GlnK (orange; PDB:6YC7), M. jannaschii GlnK with ATP (violet; PDB:2J9C), and S. elongatus GlnB complexed with NAGK (red; PDB:2V5H). In some cases, the distal part of the T-loop is disordered, and therefore not resolved.View Large Image Figure ViewerDownload Hi-res image Download (PPT)For a long time it was generally accepted that the basic mode of PII-effector molecule binding was through sensing of 2-OG in conjunction with ATP [3.Fokina O. et al.Mechanism of 2-oxoglutarate signaling by the Synechococcus elongatus PII signal transduction protein.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 19760-19765Crossref PubMed Scopus (98) Google Scholar,4.Truan D. et al.A new P(II) protein structure identifies the 2-oxoglutarate binding site.J. Mol. Biol. 2010; 400: 531-539Crossref PubMed Scopus (66) Google Scholar], with 2-OG being a status reporter for the balance of carbon and nitrogen metabolism [8.Forchhammer K. Selim K.A. Carbon/nitrogen homeostasis control in cyanobacteria.FEMS Microbiol. Rev. 2020; 44: 33-53Crossref PubMed Scopus (69) Google Scholar,9.Ninfa A.J. Jiang P. PII signal transduction proteins: sensors of alpha-ketoglutarate that regulate nitrogen metabolism.Curr. Opin. Microbiol. 2005; 8: 168-173Crossref PubMed Scopus (204) Google Scholar]. Further studies showed that ADP can also compete for the ATP-binding sites of PII [29.Zeth K. et al.Structural basis and target-specific modulation of ADP sensing by the Synechococcus elongatus PII signaling protein.J. Bio