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• W2021952073 abstract "PII signaling proteins comprise one of the most versatile signaling devices in nature and have a highly conserved structure. In cyanobacteria, PipX and N-acetyl-l-glutamate kinase are receptors of PII signaling, and these interactions are modulated by ADP, ATP, and 2-oxoglutarate. These effector molecules bind interdependently to three anti-cooperative binding sites on the trimeric PII protein and thereby affect its structure. Here we used the PII protein from Synechococcus elongatus PCC 7942 to reveal the structural basis of anti-cooperative ADP binding. Furthermore, we clarified the mutual influence of PII-receptor interaction and sensing of the ATP/ADP ratio. The crystal structures of two forms of trimeric PII, one with one ADP bound and the other with all three ADP-binding sites occupied, revealed significant differences in the ADP binding mode: at one site (S1) ADP is tightly bound through side-chain and main-chain interactions, whereas at the other two sites (S2 and S3) the ADP molecules are only bound by main-chain interactions. In the presence of the PII-receptor PipX, the affinity of ADP to the first binding site S1 strongly increases, whereas the affinity for ATP decreases due to PipX favoring the S1 conformation of PII-ADP. In consequence, the PII-PipX interaction is highly sensitive to subtle fluctuations in the ATP/ADP ratio. By contrast, the PII-N-acetyl-l-glutamate kinase interaction, which is negatively affected by ADP, is insensitive to these fluctuations. Modulation of the metabolite-sensing properties of PII by its receptors allows PII to differentially perceive signals in a target-specific manner and to perform multitasking signal transduction. PII signaling proteins comprise one of the most versatile signaling devices in nature and have a highly conserved structure. In cyanobacteria, PipX and N-acetyl-l-glutamate kinase are receptors of PII signaling, and these interactions are modulated by ADP, ATP, and 2-oxoglutarate. These effector molecules bind interdependently to three anti-cooperative binding sites on the trimeric PII protein and thereby affect its structure. Here we used the PII protein from Synechococcus elongatus PCC 7942 to reveal the structural basis of anti-cooperative ADP binding. Furthermore, we clarified the mutual influence of PII-receptor interaction and sensing of the ATP/ADP ratio. The crystal structures of two forms of trimeric PII, one with one ADP bound and the other with all three ADP-binding sites occupied, revealed significant differences in the ADP binding mode: at one site (S1) ADP is tightly bound through side-chain and main-chain interactions, whereas at the other two sites (S2 and S3) the ADP molecules are only bound by main-chain interactions. In the presence of the PII-receptor PipX, the affinity of ADP to the first binding site S1 strongly increases, whereas the affinity for ATP decreases due to PipX favoring the S1 conformation of PII-ADP. In consequence, the PII-PipX interaction is highly sensitive to subtle fluctuations in the ATP/ADP ratio. By contrast, the PII-N-acetyl-l-glutamate kinase interaction, which is negatively affected by ADP, is insensitive to these fluctuations. Modulation of the metabolite-sensing properties of PII by its receptors allows PII to differentially perceive signals in a target-specific manner and to perform multitasking signal transduction. The PII signal transduction system responds to nitrogen, carbon, and energy of the cell and is highly conserved and widespread in all three domains of life (1.Ninfa A.J. Jiang P. PII signal transduction proteins. Sensors of α-ketoglutarate that regulate nitrogen metabolism.Curr. Opin. Microbiol. 2005; 8: 168-173Crossref PubMed Scopus (207) Google Scholar, 2.Leigh J.A. Dodsworth J.A. Nitrogen regulation in bacteria and archaea.Annu. Rev. Microbiol. 2007; 61: 349-377Crossref PubMed Scopus (270) Google Scholar, 3.Forchhammer K. PII signal transducers. Novel functional and structural insights.Trends Microbiol. 2008; 16: 65-72Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 4.Sant'Anna F.H. Trentini D.B. de Souto Weber S. Cecagno R. da Silva S.C. Schrank I.S. The PII superfamily revised. A novel group and evolutionary insights.J. Mol. Evol. 2009; 68: 322-336Crossref PubMed Scopus (63) Google Scholar, 5.Huergo L.F. Pedrosa F.O. Muller-Santos M. Chubatsu L.S. Monteiro R.A. Merrick M. Souza E.M. PII signal transduction proteins. Pivotal players in post-translational control of nitrogenase activity.Microbiology. 2012; 158: 176-190Crossref PubMed Scopus (54) Google Scholar). PII proteins receive metabolic information by binding ATP, ADP, and 2-oxoglutarate (2-OG) 4The abbreviations used are: 2-OG2-oxoglutarateNAGKN-acetyl-l-glutamate kinaseSPRsurface plasmon resonanceRUresonance unitNTAnitrilotriacetic acidr.m.s.d.root mean square deviationFC2 and FC3flow cell 2 and 3, respectivelyPDBProtein data bank. and integrate the signal through conformational changes and covalent modifications (1.Ninfa A.J. Jiang P. PII signal transduction proteins. Sensors of α-ketoglutarate that regulate nitrogen metabolism.Curr. Opin. Microbiol. 2005; 8: 168-173Crossref PubMed Scopus (207) Google Scholar, 3.Forchhammer K. PII signal transducers. Novel functional and structural insights.Trends Microbiol. 2008; 16: 65-72Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Depending on the signal, PII proteins control regulatory and metabolic enzymes, transport proteins, and transcription factors involved in central nitrogen metabolism (5.Huergo L.F. Pedrosa F.O. Muller-Santos M. Chubatsu L.S. Monteiro R.A. Merrick M. Souza E.M. PII signal transduction proteins. Pivotal players in post-translational control of nitrogenase activity.Microbiology. 2012; 158: 176-190Crossref PubMed Scopus (54) Google Scholar). 2-oxoglutarate N-acetyl-l-glutamate kinase surface plasmon resonance resonance unit nitrilotriacetic acid root mean square deviation flow cell 2 and 3, respectively Protein data bank. PII proteins are homotrimers of 12–13-kDa subunits with a cylindrical body and three flexible T-loops that extend into the solvent and take part in binding of effector molecules and protein-protein interactions (3.Forchhammer K. PII signal transducers. Novel functional and structural insights.Trends Microbiol. 2008; 16: 65-72Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 5.Huergo L.F. Pedrosa F.O. Muller-Santos M. Chubatsu L.S. Monteiro R.A. Merrick M. Souza E.M. PII signal transduction proteins. Pivotal players in post-translational control of nitrogenase activity.Microbiology. 2012; 158: 176-190Crossref PubMed Scopus (54) Google Scholar). Each subunit also has two smaller loops, the B- and C-loops, that face each other in the intersubunit clefts and participate in effector binding. The PII trimer thus contains three effector nucleotide-binding sites, one in each intersubunit cleft (6.Xu Y. Carr P.D. Clancy P. Garcia-Dominguez M. Forchhammer K. Florencio F. Vasudevan S.G. Tandeau de Marsac N. Ollis D.L. The structures of the PII proteins from the cyanobacteria Synechococcus sp. PCC 7942 and Synechocystis sp. PCC 6803.Acta Crystallogr. D. 2003; 59: 2183-2190Crossref PubMed Scopus (45) Google Scholar, 7.Xu Y. Cheah E. Carr P.D. van Heeswijk W.C. Westerhoff H.V. Vasudevan S.G. Ollis D.L. GlnK, a PII-homologue. Structure reveals ATP binding site and indicates how the T-loops may be involved in molecular recognition.J. Mol. Biol. 1998; 282: 149-165Crossref PubMed Scopus (135) Google Scholar, 8.Sakai H. Wang H. Takemoto-Hori C. Kaminishi T. Yamaguchi H. Kamewari Y. Terada T. Kuramitsu S. Shirouzu M. Yokoyama S. Crystal structures of the signal transducing protein GlnK from Thermus thermophilus HB8.J. Struct. Biol. 2005; 149: 99-110Crossref PubMed Scopus (34) Google Scholar), where the adenylyl nucleotides ATP and ADP compete for the same sites. In the PII protein from the cyanobacterium Synechococcus elongatus PCC 7942, the binding of ATP and ADP to the three sites is anti-cooperative, with ATP having a higher affinity than ADP (9.Fokina O. Chellamuthu V.R. Zeth K. Forchhammer K. A novel signal transduction protein PII variant from Synechococcus elongatus PCC 7942 indicates a two-step process for NAGK-PII complex formation.J. Mol. Biol. 2010; 399: 410-421Crossref PubMed Scopus (36) Google Scholar). When Mg2+-ATP is ligated to a binding site of bacterial PII, the site can also bind 2-OG, whereas ADP binding does not support the binding of 2-OG (10.Jiang P. Ninfa A.J. Escherichia coli PII signal transduction protein controlling nitrogen assimilation acts as a sensor of adenylate energy charge in vitro.Biochemistry. 2007; 46: 12979-12996Crossref PubMed Scopus (84) Google Scholar, 11.Fokina O. Chellamuthu V.R. Forchhammer K. Zeth K. 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 (100) Google Scholar). The 2-OG binding site is located at the base of the T-loop in direct vicinity of the β- and γ-phosphate of ATP, which ligates 2-OG through a chelated Mg2+ ion (11.Fokina O. Chellamuthu V.R. Forchhammer K. Zeth K. 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 (100) Google Scholar, 12.Truan D. Huergo L.F. Chubatsu L.S. Merrick M. Li X.D. Winkler F.K. A new PII protein structure identifies the 2-oxoglutarate binding site.J. Mol. Biol. 2010; 400: 531-539Crossref PubMed Scopus (66) Google Scholar). Consequently, binding of ATP and 2-OG is synergistic (13.Forchhammer K. Hedler A. Phosphoprotein PII from cyanobacteria - analysis of functional conservation with the PII signal-transduction protein from Escherichia coli.Eur. J. Biochem. 1997; 244: 869-875Crossref PubMed Scopus (80) Google Scholar). Because ADP competes with Mg2+-ATP in binding to PII, it negatively affects the binding of 2-OG and thereby interferes with the ability of PII to sense the signal of nitrogen starvation (10.Jiang P. Ninfa A.J. Escherichia coli PII signal transduction protein controlling nitrogen assimilation acts as a sensor of adenylate energy charge in vitro.Biochemistry. 2007; 46: 12979-12996Crossref PubMed Scopus (84) Google Scholar, 14.Radchenko M.V. Thornton J. Merrick M. Control of AmtB-GlnK complex formation by intracellular levels of ATP, ADP, and 2-oxoglutarate.J. Biol. Chem. 2010; 285: 31037-31045Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 15.Fokina O. Herrmann C. Forchhammer K. Signal-transduction protein PII from Synechococcus elongatus PCC 7942 senses low adenylate energy charge in vitro.Biochem. J. 2011; 440: 147-156Crossref PubMed Scopus (30) Google Scholar). The three 2-OG binding sites exhibit negative cooperativity relative to each other, which is mediated through intersubunit signaling inside the trimer (9.Fokina O. Chellamuthu V.R. Zeth K. Forchhammer K. A novel signal transduction protein PII variant from Synechococcus elongatus PCC 7942 indicates a two-step process for NAGK-PII complex formation.J. Mol. Biol. 2010; 399: 410-421Crossref PubMed Scopus (36) Google Scholar, 11.Fokina O. Chellamuthu V.R. Forchhammer K. Zeth K. 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 (100) Google Scholar). In living cells ATP and ADP are simultaneously present, and they compete for the three nucleotide-binding sites of the PII trimer. The total concentration of these effectors in bacterial cells is in the millimolar range (16.Chapman A.G. Fall L. Atkinson D.E. Adenylate energy charge in Escherichia coli during growth and starvation.J. Bacteriol. 1971; 108: 1072-1086Crossref PubMed Google Scholar, 17.Chapman A.G. Atkinson D.E. Adenine nucleotide concentrations and turnover rates. Their correlation with biological activity in bacteria and yeast.Adv. Microb. Physiol. 1977; 15: 253-306Crossref PubMed Scopus (188) Google Scholar, 18.Kallas T. Castenholz R.W. Internal pH and ATP-ADP pools in the cyanobacterium Synechococcus sp. during exposure to growth-inhibiting low pH.J. Bacteriol. 1982; 149: 229-236Crossref PubMed Google Scholar), well above the KD for the PII binding sites (9.Fokina O. Chellamuthu V.R. Zeth K. Forchhammer K. A novel signal transduction protein PII variant from Synechococcus elongatus PCC 7942 indicates a two-step process for NAGK-PII complex formation.J. Mol. Biol. 2010; 399: 410-421Crossref PubMed Scopus (36) Google Scholar); therefore, all three binding sites of PII are presumably always occupied by either ATP or ADP. Considering that 2-OG is only able to bind to the sites occupied by Mg2+-ATP, PII proteins could theoretically adopt 20 different ligand binding states, but considering that in vivo all sites are filled with either ATP or ADP, only 10 states would be possible. If the ligand binding properties are taken into account, only 5–7 states are probable in vivo. At high ATP concentrations, all PII sites are filled with Mg-ATP (PII-ATP3) and up to three 2-OG molecules can be bound, thereby measuring this effector with the highest sensitivity. As the ADP concentration increases, the PII-ATP2-ADP1 population forms. Only when the ADP concentration exceeds that of ATP would the PII-ATP1-ADP2 population accumulate, as the binding affinity of ADP is lower than that of ATP. In general, the mixed populations (PII-ATP2-ADP1 and PII-ATP1-ADP2) have fewer 2-OG binding sites than PII-ATP3, and calorimetric titration experiments have shown that at an ATP:ADP ratio of 1:1, only one 2-OG is bound (15.Fokina O. Herrmann C. Forchhammer K. Signal-transduction protein PII from Synechococcus elongatus PCC 7942 senses low adenylate energy charge in vitro.Biochem. J. 2011; 440: 147-156Crossref PubMed Scopus (30) Google Scholar). The failure to detect two binding sites for 2-OG under these conditions indicates either that the PII-ATP2-ADP1 species can bind only one 2-OG molecule or, more unlikely, that in the presence of equimolar ADP and ATP, the PII-ATP1-ADP2 species is preferentially formed. PII proteins from oxygenic phototrophs (cyanobacteria and plants) control arginine biosynthesis through the arginine feedback-inhibited enzyme N-acetyl-l-glutamate kinase (NAGK) (19.Burillo S. Luque I. Fuentes I. Contreras A. Interactions between the nitrogen signal transduction protein PII and N-acetyl glutamate kinase in organisms that perform oxygenic photosynthesis.J. Bacteriol. 2004; 186: 3346-3354Crossref PubMed Scopus (102) Google Scholar, 20.Heinrich A. Maheswaran M. Ruppert U. Forchhammer K. The Synechococcus elongatus PII signal transduction protein controls arginine synthesis by complex formation with N-acetyl-l-glutamate kinase.Mol. Microbiol. 2004; 52: 1303-1314Crossref PubMed Scopus (109) Google Scholar). PII binding to NAGK enhances the activity of the enzyme and protects it from feedback inhibition by arginine (21.Maheswaran M. Urbanke C. Forchhammer K. Complex formation and catalytic activation by the PII signaling protein of N-acetyl-l-glutamate kinase from Synechococcus elongatus strain PCC 7942.J. Biol. Chem. 2004; 279: 55202-55210Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). PII in the ATP-ligated state avidly binds to NAGK, whereas in the presence of ADP, complex formation is impaired, and as a consequence, NAGK activity is not enhanced (15.Fokina O. Herrmann C. Forchhammer K. Signal-transduction protein PII from Synechococcus elongatus PCC 7942 senses low adenylate energy charge in vitro.Biochem. J. 2011; 440: 147-156Crossref PubMed Scopus (30) Google Scholar). Likewise, the effector 2-OG strongly inhibits PII-NAGK complex formation. Thus, the inhibitory effect of 2-OG is not antagonized by ADP and is, therefore, robust against different ATP/ADP ratios (15.Fokina O. Herrmann C. Forchhammer K. Signal-transduction protein PII from Synechococcus elongatus PCC 7942 senses low adenylate energy charge in vitro.Biochem. J. 2011; 440: 147-156Crossref PubMed Scopus (30) Google Scholar, 21.Maheswaran M. Urbanke C. Forchhammer K. Complex formation and catalytic activation by the PII signaling protein of N-acetyl-l-glutamate kinase from Synechococcus elongatus strain PCC 7942.J. Biol. Chem. 2004; 279: 55202-55210Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). In addition to the arginine pathway control, cyanobacterial PII proteins indirectly control gene expression by sequestering PipX, which is a co-activator of the global nitrogen control transcription factor NtcA (22.Espinosa J. Forchhammer K. Burillo S. Contreras A. Interaction network in cyanobacterial nitrogen regulation. PipX, a protein that interacts in a 2-oxoglutarate dependent manner with PII and NtcA.Mol. Microbiol. 2006; 61: 457-469Crossref PubMed Scopus (128) Google Scholar, 23.Espinosa J. Forchhammer K. Contreras A. Role of the Synechococcus PCC 7942 nitrogen regulator protein PipX in NtcA-controlled processes.Microbiology. 2007; 153: 711-718Crossref PubMed Scopus (57) Google Scholar, 24.Llácer J.L. Espinosa J. Castells M.A. Contreras A. Forchhammer K. Rubio V. Structural basis for the regulation of NtcA-dependent transcription by proteins PipX and PII.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 15397-15402Crossref PubMed Scopus (96) Google Scholar). PII binds PipX in the absence of 2-OG, with ADP strongly enhancing the PII-PipX interaction and antagonizing the inhibitory 2-OG effect even at low ADP concentrations (15.Fokina O. Herrmann C. Forchhammer K. Signal-transduction protein PII from Synechococcus elongatus PCC 7942 senses low adenylate energy charge in vitro.Biochem. J. 2011; 440: 147-156Crossref PubMed Scopus (30) Google Scholar, 24.Llácer J.L. Espinosa J. Castells M.A. Contreras A. Forchhammer K. Rubio V. Structural basis for the regulation of NtcA-dependent transcription by proteins PipX and PII.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 15397-15402Crossref PubMed Scopus (96) Google Scholar). Binding of ATP and ADP is a general property of PII proteins in living cells (5.Huergo L.F. Pedrosa F.O. Muller-Santos M. Chubatsu L.S. Monteiro R.A. Merrick M. Souza E.M. PII signal transduction proteins. Pivotal players in post-translational control of nitrogenase activity.Microbiology. 2012; 158: 176-190Crossref PubMed Scopus (54) Google Scholar). In Escherichia coli, which has two PII paralogues, GlnB and GlnK, ADP acts as an antagonist of 2-OG in the GlnB-mediated regulation of the NRI/NRII two-component system and of the adenylyltransferase-glutamine synthetase monocycle in vitro. ADP also inhibits uridylylation of GlnB by the bifunctional enzyme uridylyltransferase/uridylyl-removing enzyme (UTase) (10.Jiang P. Ninfa A.J. Escherichia coli PII signal transduction protein controlling nitrogen assimilation acts as a sensor of adenylate energy charge in vitro.Biochemistry. 2007; 46: 12979-12996Crossref PubMed Scopus (84) Google Scholar, 25.Jiang P. Ninfa A.J. α-Ketoglutarate controls the ability of the Escherichia coli PII signal transduction protein to regulate the activities of NRII (NtrB) but does not control the binding of PII to NRII.Biochemistry. 2009; 48: 11514-11521Crossref PubMed Scopus (31) Google Scholar). In the presence of ADP, the PII paralogue GlnK forms a complex and thereby blocks the ammonium transporter AmtB, a reaction that is antagonized by Mg2+/ATP/2-OG (14.Radchenko M.V. Thornton J. Merrick M. Control of AmtB-GlnK complex formation by intracellular levels of ATP, ADP, and 2-oxoglutarate.J. Biol. Chem. 2010; 285: 31037-31045Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Recently, Radchenko et al. (26.Radchenko M.V. Thornton J. Merrick M. PII signal transduction proteins are ATPases whose activity is regulated by 2-oxoglutarate.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 12948-12953Crossref PubMed Scopus (38) Google Scholar) showed that in the absence of 2-OG, GlnK displays intrinsic ATPase activity, slowly converting ATP-GlnK to ADP-GlnK, which then tightly binds AmtB. Sensing of the energy charge levels has also been suggested for the PII proteins from Rhodospirillum rubrum (27.Zhang Y. Pohlmann E.L. Halbleib C.M. Ludden P.W. Roberts G.P. Effect of PII and its homolog GlnK on reversible ADP-ribosylation of dinitrogenase reductase by heterologous expression of the Rhodospirillum rubrum dinitrogenase reductase ADP-ribosyl transferase-dinitrogenase reductase-activating glycohydrolase regulatory system in Klebsiella pneumoniae.J. Bacteriol. 2001; 183: 1610-1620Crossref PubMed Scopus (29) Google Scholar, 28.Zhang Y.P. Pohlmann E.L. Ludden P.W. Roberts G.P. Regulation of nitrogen fixation by multiple PII homologs in the photosynthetic bacterium Rhodospirillum rubrum.Symbiosis. 2003; 35: 85-100Google Scholar). ADP stabilizes the GlnJ-AmtB1 complex (GlnJ is a GlnK paralogue) even in the presence of complex-dissociating ATP-2-OG levels (29.Teixeira P.F. Jonsson A. Frank M. Wang H. Nordlund S. Interaction of the signal transduction protein GlnJ with the cellular targets AmtB1, GlnE and GlnD in Rhodospirillum rubrum. Dependence on manganese, 2-oxoglutarate, and the ADP/ATP ratio.Microbiology. 2008; 154: 2336-2347Crossref PubMed Scopus (25) Google Scholar). The affinities toward ATP and ADP and the anti-cooperativity between the binding sites are key features of PII proteins that determine their ability to function as energy sensors. In this respect it is important to note that the fine-tuned ligand binding properties of PII proteins from different sources are variable (30.Maier S. Schleberger P. Lü W. Wacker T. Pflüger T. Litz C. Andrade S.L. Mechanism of disruption of the Amt-GlnK complex by PII-mediated sensing of 2-oxoglutarate.PLoS ONE. 2011; 6: e26327Crossref PubMed Scopus (28) Google Scholar). One of the best-studied PII proteins is that of S. elongatus. Many structure-function details related to 2-OG signaling have been elucidated (3.Forchhammer K. PII signal transducers. Novel functional and structural insights.Trends Microbiol. 2008; 16: 65-72Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 11.Fokina O. Chellamuthu V.R. Forchhammer K. Zeth K. 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 (100) Google Scholar, 24.Llácer J.L. Espinosa J. Castells M.A. Contreras A. Forchhammer K. Rubio V. Structural basis for the regulation of NtcA-dependent transcription by proteins PipX and PII.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 15397-15402Crossref PubMed Scopus (96) Google Scholar, 31.Zeth K. Fokina O. Forchhammer K. 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) (an overview of the various S. elongatus PII structures is presented in Table 1). However, the structural implications of ADP binding and the functional consequences for the integrated NAGK-PII·PipX interaction network remain elusive.TABLE 1Overview of structural data used in this workStructure PDB entryProtein(s) and organismNumber of subunits in the AUDetailed description of AUDetailed co-factors descriptionNomenclature of the PII trimer in this paperNomenclature of individual effector sites in this paperResolution (Å)Reference4C3LPII fromOne monomerNonePIINCI1.6This workS. elongatus4C3MPII fromOne trimerNonePIINCII2.2This workS. elongatus4C3KPII fromTwo trimersPIIADP3.1This workS. elongatusTrimer IOne ADPPIIADP1PIIADP1-S1Trimer IIThree ADPPIIADP3PIIADP3-S1PIIADP3-S2PIIADP3-S32XZWPII from S. elongatusThree trimersTrimer IThree ATPPIIOG1PIIOG1-S11.95(11.Fokina O. Chellamuthu V.R. Forchhammer K. Zeth K. 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 (100) Google Scholar)One Mg2+PIIOG1-S2One 2-OGPIIOG1-S3Trimer IIThree ATPPIIOG2PIIOG2-S1Two Mg2+PIIOG2-S2Two 2-OGPIIOG2-S3Trimer IIIThree ATPPIIOG3PIIOG3-S1Three Mg2+PIIOG3-S2Three 2-OGPIIOG3-S32XULPII from S. elongatusTwo identical trimersThree ATPPIIOGex2.2(11.Fokina O. Chellamuthu V.R. Forchhammer K. Zeth K. 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 (100) Google Scholar)Three Mg2+Three 2-OG2XBPPII from S. elongatus (I86N mutant)One monomerOne ATP1.2(9.Fokina O. Chellamuthu V.R. Zeth K. Forchhammer K. A novel signal transduction protein PII variant from Synechococcus elongatus PCC 7942 indicates a two-step process for NAGK-PII complex formation.J. Mol. Biol. 2010; 399: 410-421Crossref PubMed Scopus (36) Google Scholar)One Mg2+4AFFPII from S. elongatus (I86N mutant)One monomerOne ATP1.05(31.Zeth K. Fokina O. Forchhammer K. 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)One Mg2+One citrate1QY7PII from S. elongatus (S49A mutant)One trimerNone2.0(6.Xu Y. Carr P.D. Clancy P. Garcia-Dominguez M. Forchhammer K. Florencio F. Vasudevan S.G. Tandeau de Marsac N. Ollis D.L. The structures of the PII proteins from the cyanobacteria Synechococcus sp. PCC 7942 and Synechocystis sp. PCC 6803.Acta Crystallogr. D. 2003; 59: 2183-2190Crossref PubMed Scopus (45) Google Scholar)2J9DGlnK1 from M. jannashiiFour trimersTrimer I Trimer II Trimer III Trimer IVOne ADP One AMP One ADP Two ADP2.1(42.Yildiz O. Kalthoff C. Raunser S. Kühlbrandt W. Structure of GlnK1 with bound effectors indicates regulatory mechanism for ammonia uptake.EMBO J. 2007; 26: 589-599Crossref PubMed Scopus (55) Google Scholar)1V9OPII from T. thermophilusOne trimerThree ADP(8.Sakai H. Wang H. Takemoto-Hori C. Kaminishi T. Yamaguchi H. Kamewari Y. Terada T. Kuramitsu S. Shirouzu M. Yokoyama S. Crystal structures of the signal transducing protein GlnK from Thermus thermophilus HB8.J. Struct. Biol. 2005; 149: 99-110Crossref PubMed Scopus (34) Google Scholar)3TA1PII from A, fulgidusOne trimerThree ADP1.9(30.Maier S. Schleberger P. Lü W. Wacker T. Pflüger T. Litz C. Andrade S.L. Mechanism of disruption of the Amt-GlnK complex by PII-mediated sensing of 2-oxoglutarate.PLoS ONE. 2011; 6: e26327Crossref PubMed Scopus (28) Google Scholar)3NCRPII from A. fulgidusOne trimerThree ADP1.44(41.Helfmann S. Lü W. Litz C. Andrade S.L. Cooperative Binding of MgATP and MgADP in the Trimeric PII Protein GlnK2 from Archaeoglobus fulgidus.J. Mol. Biol. 2010; 402: 165-177Crossref PubMed Scopus (26) Google Scholar)2XG8PII/PipX from S. elongatusOne trimeric complexNone3.2(24.Llácer J.L. Espinosa J. Castells M.A. Contreras A. Forchhammer K. Rubio V. Structural basis for the regulation of NtcA-dependent transcription by proteins PipX and PII.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 15397-15402Crossref PubMed Scopus (96) Google Scholar)3N5BPII/PipX from Nostoc sp.One monomeric complexADP1.9(48.Zhao M.X. Jiang Y.L. Xu B.Y. Chen Y. Zhang C.C. Zhou C.Z. Crystal structure of the cyanobacterial signal transduction protein PII in complex with PipX.J. Mol. Biol. 2010; 402: 552-559Crossref PubMed Scopus (32) Google Scholar)2V5HPII/NAGK from S. elongatusOne NAGK hexamer two PII trimersN-Acetyl-l-glutamate (bound to NAGK)2.75(46.Llácer J.L. Contreras A. Forchhammer K. Marco-Marín C. Gil-Ortiz F. Maldonado R. Fita I. Rubio V. The crystal structure of the complex of PII and acetylglutamate kinase reveals how PII controls the storage of nitrogen as arginine.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 17644-17649Crossref PubMed Scopus (97) Google Scholar) Open table in a new tab In the present study we investigated the structural transitions of effector-free and ADP-loaded PII subunits from S. elongatus and propose a mechanism of ADP sensing by PII. We also analyzed the role of energy charge sensing (energy charge measured as the ATP/ADP ratio) for the PipX/NtcA-PII·NAGK interaction network. Based on our data and previously published data, we conclude that the PII signal transduction system does not work as an on/off switch governed by the intrinsic ATPase activity of PI, but rather as a multitasking signal integration device with flexible signal output dependent on its receptor protein. The glnB and pipX genes from S. elongatus, cloned into the Strep-tag fusion vector pASK-IBA3 (IBA, Göttingen, Germany), were overexpressed in E. coli RB9060 (32.Bueno R. Pahel G. Magasanik B. Role of glnB and glnD gene products in regulation of the glnALG operon of Escherichia coli.J. Bacteriol. 1985; 164: 816-822Crossref PubMed Google Scholar), and the PII and PipX proteins, each with a C-terminal fused Strep-tag peptide, were purified using affinity chromatography as described previously (20.Heinrich A. Maheswaran M. Ruppert U. Forchhammer K. The Synechococcus elongatus PII signal transduction protein controls arginine synthesis by complex formation with N-acetyl-l-glutamate kinase.Mol. Microbiol. 2004; 52: 1303-1314Crossref PubMed Scopus (109) Google Scholar, 22.Espinosa J. Forchhammer K. Burillo S. Contreras A. Interaction network in cyanobacterial nitrogen regulation. PipX, a protein that interacts in a 2-oxoglutarate dependent manner with PII and NtcA.Mol. Microbiol. 2006; 61: 457-469Crossref PubMed Scopus (128) Google Scholar). The genes encoding NAGK and NtcA from S. elongatus, cloned in pET15b vector, were overexpressed in E. coli strain BL21(DE3) (33.Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Use of T7 RNA polymerase to direct expression of cl" @default.
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