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• W2051200740 abstract "The activity of the nitrogenase enzyme in the diazotroph Azospirillum brasilense is reversibly inactivated by ammonium through ADP-ribosylation of the nitrogenase NifH subunit. This process is catalyzed by DraT and is reversed by DraG, and the activities of both enzymes are regulated according to the levels of ammonium through direct interactions with the PII proteins GlnB and GlnZ. We have previously shown that DraG interacts with GlnZ both in vivo and in vitro and that DraT interacts with GlnB in vivo. We have now characterized the influence of PII uridylylation status and the PII effectors (ATP, ADP, and 2-oxoglutarate) on the in vitro formation of DraT-GlnB and DraG-GlnZ complexes. We observed that both interactions are maximized when PII proteins are de-uridylylated and when ADP is present. The DraT-GlnB complex formed in vivo was purified to homogeneity in the presence of ADP. The stoichiometry of the DraT-GlnB complex was determined by three independent approaches, all of which indicated a 1:1 stoichiometry (DraT monomer:GlnB trimer). Our results suggest that the intracellular fluctuation of the PII ligands ATP, ADP, and 2-oxoglutarate play a key role in the post-translational regulation of nitrogenase activity. The activity of the nitrogenase enzyme in the diazotroph Azospirillum brasilense is reversibly inactivated by ammonium through ADP-ribosylation of the nitrogenase NifH subunit. This process is catalyzed by DraT and is reversed by DraG, and the activities of both enzymes are regulated according to the levels of ammonium through direct interactions with the PII proteins GlnB and GlnZ. We have previously shown that DraG interacts with GlnZ both in vivo and in vitro and that DraT interacts with GlnB in vivo. We have now characterized the influence of PII uridylylation status and the PII effectors (ATP, ADP, and 2-oxoglutarate) on the in vitro formation of DraT-GlnB and DraG-GlnZ complexes. We observed that both interactions are maximized when PII proteins are de-uridylylated and when ADP is present. The DraT-GlnB complex formed in vivo was purified to homogeneity in the presence of ADP. The stoichiometry of the DraT-GlnB complex was determined by three independent approaches, all of which indicated a 1:1 stoichiometry (DraT monomer:GlnB trimer). Our results suggest that the intracellular fluctuation of the PII ligands ATP, ADP, and 2-oxoglutarate play a key role in the post-translational regulation of nitrogenase activity. Biological nitrogen fixation is catalyzed by the nitrogenase enzyme and is a very energy-demanding process requiring at least 16 mol of ATP per mol of N2 reduced. Consequently it is tightly regulated both at the transcriptional and, in some cases, at the post-translational level (1Dixon R. Kahn D. Nat. Rev. Microbiol. 2004; 2: 621-631Crossref PubMed Scopus (729) Google Scholar). In some free-living bacteria, nitrogenase is down-regulated at the post-translational level by reversible ADP-ribosylation of the nitrogenase Fe-protein or NifH. This modification is catalyzed by dinitrogenase reductase ADP-ribosyltransferase (DraT) 3The abbreviations used are: DraT, dinitrogenase reductase ADP-ribosyltransferase; DraG, dinitrogenase reductase-activating glycohydrolase; 2-OG, 2-oxoglutarate; LDAO, lauryldimethylamine oxide; GS, glutamine synthetase; GOGAT, glutamate synthase. and is reversed by dinitrogenase reductase-activating glycohydrolase (DraG) (2Nordlund S. Triplett E.W. Prokaryotic Nitrogen Fixation: A Model System for Analysis of Biological Processes. Horizon Scientific Press, Wymondham, UK2000: 149-167Google Scholar, 3Zhang Y. Burris R.H. Ludden P.W. Roberts G.P. FEMS Microbiol. Lett. 1997; 152: 195-204Crossref PubMed Google Scholar). Nitrogenase inactivation is induced by ammonium in the external medium or by a decrease in the energy charge of the cell, the main component of which is the ATP/ADP ratio (4Chapman A.G. Fall L. Atkinson D.E. J. Bacteriol. 1971; 108: 1072-1086Crossref PubMed Google Scholar, 5Upchurch R.G. Mortenson L.E. J. Bacteriol. 1980; 143: 274-284Crossref PubMed Google Scholar). In Azospirillum brasilense, the latter response occurs upon an anaerobic shift whereas in Rhodospirillum rubrum and Rhodobacter capsulatus it occurs in response to darkness (2Nordlund S. Triplett E.W. Prokaryotic Nitrogen Fixation: A Model System for Analysis of Biological Processes. Horizon Scientific Press, Wymondham, UK2000: 149-167Google Scholar, 3Zhang Y. Burris R.H. Ludden P.W. Roberts G.P. FEMS Microbiol. Lett. 1997; 152: 195-204Crossref PubMed Google Scholar). The model for DraT and DraG regulation suggests that DraT is inactive and DraG is active before the cell is exposed to a negative stimulus for nitrogenase (3Zhang Y. Burris R.H. Ludden P.W. Roberts G.P. FEMS Microbiol. Lett. 1997; 152: 195-204Crossref PubMed Google Scholar). When ammonium is added to the medium, DraT is transiently activated and DraG is inactivated leading to NifH modification and inactivation. The DraG enzyme is reactivated as the negative stimulus is exhausted, causing NifH re-activation (3Zhang Y. Burris R.H. Ludden P.W. Roberts G.P. FEMS Microbiol. Lett. 1997; 152: 195-204Crossref PubMed Google Scholar). Recently, several features of the ammonium-signaling pathway that control DraT and DraG activities have begun to be understood (6Tremblay P.L. Drepper T. Masepohl B. Hallenbeck P.C. J. Bacteriol. 2007; 189: 5850-5859Crossref PubMed Scopus (22) Google Scholar, 7Tremblay P.L. Hallenbeck P.C. J. Bacteriol. 2008; 190: 1588-1594Crossref PubMed Scopus (17) Google Scholar, 8Zhang Y. Wolfe D.M. Pohlmann E.L. Conrad M.C. Roberts G.P. Microbiology. 2006; 152: 2075-2089Crossref PubMed Scopus (30) Google Scholar, 9Wang H. Franke C.C. Nordlund S. Noren A. FEMS Microbiol. Lett. 2005; 253: 273-279Crossref PubMed Scopus (35) Google Scholar, 10Huergo L.F. Chubatsu L.S. Souza E.M. Pedrosa F.O. Steffens M.B. Merrick M. FEBS Lett. 2006; 580: 5232-5236Crossref PubMed Scopus (36) Google Scholar, 11Huergo L.F. Souza E.M. Araujo M.S. Pedrosa F.O. Chubatsu L.S. Steffens M.B. Merrick M. Mol. Microbiol. 2006; 59: 326-337Crossref PubMed Scopus (56) Google Scholar, 12Huergo L.F. Merrick M. Pedrosa F.O. Chubatsu L.S. Araujo L.M. Souza E.M. Mol. Microbiol. 2007; 66: 1523-1535PubMed Google Scholar). In all cases, regulation of DraT and DraG appears to involve direct interaction between these enzymes and the nitrogen-signaling PII proteins. In A. brasilense, which encodes two PII proteins, GlnB and GlnZ (13de Zamaroczy M. Mol. Microbiol. 1998; 29: 449-463Crossref PubMed Scopus (70) Google Scholar), a model for this process has recently been proposed (10Huergo L.F. Chubatsu L.S. Souza E.M. Pedrosa F.O. Steffens M.B. Merrick M. FEBS Lett. 2006; 580: 5232-5236Crossref PubMed Scopus (36) Google Scholar). Upon an ammonium shock, GlnB interacts with DraT, and this is presumed to trigger DraT activation. GlnZ interacts with both DraG and the ammonium transporter AmtB on the cell membrane. The formation of a ternary complex between AmtB, GlnZ, and DraG efficiently targets DraG to the cell membrane where it is presumed to be catalytically inactive (10Huergo L.F. Chubatsu L.S. Souza E.M. Pedrosa F.O. Steffens M.B. Merrick M. FEBS Lett. 2006; 580: 5232-5236Crossref PubMed Scopus (36) Google Scholar, 12Huergo L.F. Merrick M. Pedrosa F.O. Chubatsu L.S. Araujo L.M. Souza E.M. Mol. Microbiol. 2007; 66: 1523-1535PubMed Google Scholar). The PII protein family is very widely distributed, and several features of these proteins have been recently reviewed (14Forchhammer K. Trends Microbiol. 2008; 16: 65-77Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 15Leigh J.A. Dodsworth J.A. Annu. Rev. Microbiol. 2007; 61: 349-377Crossref PubMed Scopus (270) Google Scholar). PII proteins are trimeric proteins that control cellular nitrogen metabolism by direct protein-protein interaction with several enzymes, transcriptional regulators, and transporters. The structures of PII proteins from several organisms have been determined, and they are in general highly conserved. The trimer consists of a core compact barrel with three protruding loops: the T, B, and C loops (16Carr P.D. Cheah E. Suffolk P.M. Vasudevan S.G. Dixon N.E. Ollis D.L. Acta Crystallogr. 1996; D52: 93-104Google Scholar, 17Xu Y. Cheah E. Carr P.D. van Heeswijk W.C. Westerhoff H.V. Vasudevan S.G. Ollis D.L. J. Mol. Biol. 1998; 282: 149-165Crossref PubMed Scopus (135) Google Scholar). PII protein activity is regulated through binding of the allosteric effectors ATP, ADP, and 2-oxoglutarate (2-OG). In many cases, these proteins are also regulated by covalent modification of the T-loop. This can involve uridylylation in Gram-negative bacteria (18Atkinson M.R. Kamberov E.S. Weiss R.L. Ninfa A.J. J. Biol. Chem. 1994; 269: 28288-28293Abstract Full Text PDF PubMed Google Scholar), adenylylation in Gram-positive bacteria (19Strosser J. Ludke A. Schaffer S. Kramer R. Burkovski A. Mol. Microbiol. 2004; 54: 132-147Crossref PubMed Scopus (86) Google Scholar), and phosphorylation in cyanobacteria (20Forchhammer K. FEMS Microbiol. Rev. 2004; 28: 319-333Crossref PubMed Scopus (203) Google Scholar). The PII proteins from Escherichia coli can bind up to three molecules of ATP and 2-OG in a positive synergistic manner (21Kamberov E.S. Atkinson M.R. Ninfa A.J. J. Biol. Chem. 1995; 270: 17797-17807Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar), the ATP-binding site being located in the lateral clefts between the subunits (17Xu Y. Cheah E. Carr P.D. van Heeswijk W.C. Westerhoff H.V. Vasudevan S.G. Ollis D.L. J. Mol. Biol. 1998; 282: 149-165Crossref PubMed Scopus (135) Google Scholar, 22Xu Y. Carr P.D. Huber T. Vasudevan S.G. Ollis D.L. Eur. J. Biochem. 2001; 268: 2028-2037Crossref PubMed Scopus (50) Google Scholar). Co-crystallization of Methanococcus jannaschii GlnK with Mg-ATP and 2-OG suggested that Mg-ATP binding to the lateral cleft causes the T-loop to assume a compact conformation that creates a 2-OG-binding site in the T-loop. In this structure, 2-OG is accommodated mainly by hydrogen bond formation with the main chain backbone (23Yildiz O. Kalthoff C. Raunser S. Kuhlbrandt W. EMBO J. 2007; 26: 589-599Crossref PubMed Scopus (55) Google Scholar). Recent data from several organisms indicate that the PII ATP-binding site can be occupied by ADP in a competitive manner, suggesting that PII proteins can act as sensors of the energy charge of the cell (12Huergo L.F. Merrick M. Pedrosa F.O. Chubatsu L.S. Araujo L.M. Souza E.M. Mol. Microbiol. 2007; 66: 1523-1535PubMed Google Scholar, 23Yildiz O. Kalthoff C. Raunser S. Kuhlbrandt W. EMBO J. 2007; 26: 589-599Crossref PubMed Scopus (55) Google Scholar, 24Jiang P. Ninfa A.J. Biochemistry. 2007; 46: 12979-12996Crossref PubMed Scopus (84) Google Scholar, 25Wolfe D.M. Zhang Y. Roberts G.P. J. Bacteriol. 2007; 189: 6861-6869Crossref PubMed Scopus (29) Google Scholar, 26Teixeira P.F. Jonsson A. Frank M. Wang H. Nordlund S. Microbiology. 2008; 154: 2336-2347Crossref PubMed Scopus (25) Google Scholar, 27Conroy M.J. Durand A. Lupo D. Li X.D. Bullough P.A. Winkler F.K. Merrick M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 1213-1218Crossref PubMed Scopus (146) Google Scholar). The A. brasilense PII proteins, GlnB and GlnZ, are subject to a cycle of uridylylation/de-uridylylation catalyzed by the bifunctional enzyme GlnD (28de Zamaroczy M. Paquelin A. Peltre G. Forchhammer K. Elmerich C. J. Bacteriol. 1996; 178: 4143-4149Crossref PubMed Google Scholar, 29van Dommelen A. Keijers V. Somers E. Vanderleyden J. Mol. Genet. Genomics. 2002; 266: 813-820Crossref PubMed Scopus (14) Google Scholar). Under nitrogen-limiting conditions GlnB and GlnZ are fully uridylylated, and upon an ammonium shock GlnD promotes their de-uridylylation (28de Zamaroczy M. Paquelin A. Peltre G. Forchhammer K. Elmerich C. J. Bacteriol. 1996; 178: 4143-4149Crossref PubMed Google Scholar, 30Araujo L.M. Huergo L.F. Invitti A.L. Gimenes C.I. Bonatto A.C. Monteiro R.A. Souza E.M. Pedrosa F.O. Chubatsu L.S. Braz. J. Med. Biol. Res. 2008; 41: 289-294Crossref PubMed Scopus (22) Google Scholar, 31Araujo M.S. Baura V.A. Souza E.M. Benelli E.M. Rigo L.U. Steffens M.B. Pedrosa F.O. Chubatsu L.S. Protein Expr. Purif. 2004; 33: 19-24Crossref PubMed Scopus (16) Google Scholar). In vivo experiments in A. brasilense have shown that the uridylylation cycle of the PII proteins in response to ammonium is synchronized with nitrogenase ADP-ribosylation and hence with changes in DraT and DraG activities (11Huergo L.F. Souza E.M. Araujo M.S. Pedrosa F.O. Chubatsu L.S. Steffens M.B. Merrick M. Mol. Microbiol. 2006; 59: 326-337Crossref PubMed Scopus (56) Google Scholar). Previous results have shown that de-uridylylated GlnZ and GlnB can interact with DraG and DraT, respectively, in A. brasilense (10Huergo L.F. Chubatsu L.S. Souza E.M. Pedrosa F.O. Steffens M.B. Merrick M. FEBS Lett. 2006; 580: 5232-5236Crossref PubMed Scopus (36) Google Scholar). In this detailed study, we have now characterized the influence of PII uridylylation status and the PII effectors on in vitro formation of DraT-GlnB and DraG-GlnZ complexes. We have also determined the stoichiometry of the DraT-GlnB complex. Protein Analysis-Electrophoresis of proteins was carried out by SDS-PAGE, and gels were Coomassie Blue-stained unless indicated otherwise. Signals on gels were quantified using the Lab Works (UVP) or the ImageQuant (GE Healthcare) software where results are reported in arbitrary units. Protein concentrations were determined by the Bradford assay using bovine serum albumin as a standard. Western blots were carried out as described, using polyclonal rabbit anti-DraT antibody (11Huergo L.F. Souza E.M. Araujo M.S. Pedrosa F.O. Chubatsu L.S. Steffens M.B. Merrick M. Mol. Microbiol. 2006; 59: 326-337Crossref PubMed Scopus (56) Google Scholar). MALDI-TOF analysis was performed as described previously (30Araujo L.M. Huergo L.F. Invitti A.L. Gimenes C.I. Bonatto A.C. Monteiro R.A. Souza E.M. Pedrosa F.O. Chubatsu L.S. Braz. J. Med. Biol. Res. 2008; 41: 289-294Crossref PubMed Scopus (22) Google Scholar). Protein Purification-The A. brasilense GlnB, GlnZ, HisGlnD, HisDraG, and HisDraT proteins were purified as described previously (12Huergo L.F. Merrick M. Pedrosa F.O. Chubatsu L.S. Araujo L.M. Souza E.M. Mol. Microbiol. 2007; 66: 1523-1535PubMed Google Scholar, 30Araujo L.M. Huergo L.F. Invitti A.L. Gimenes C.I. Bonatto A.C. Monteiro R.A. Souza E.M. Pedrosa F.O. Chubatsu L.S. Braz. J. Med. Biol. Res. 2008; 41: 289-294Crossref PubMed Scopus (22) Google Scholar, 32Huergo L.F. Souza E.M. Steffens M.B. Yates M.G. Pedrosa F.O. Chubatsu L.S. Arch. Microbiol. 2005; 183: 209-217Crossref PubMed Scopus (10) Google Scholar). For the purification of the HisDraT-GlnB complex, both proteins were co-expressed in E. coli BL21 from the plasmids pLHPETDraT (32Huergo L.F. Souza E.M. Steffens M.B. Yates M.G. Pedrosa F.O. Chubatsu L.S. Arch. Microbiol. 2005; 183: 209-217Crossref PubMed Scopus (10) Google Scholar) and pLHDK5pII (33Huergo L.F. Filipaki A. Chubatsu L.S. Yates M.G. Steffens M.B. Pedrosa F.O. Souza E.M. FEMS Microbiol. Lett. 2005; 253: 47-54Crossref PubMed Scopus (12) Google Scholar) overnight at 20 °C using isopropyl-1-thio-β-d-galactopyranoside 0.3 mm as inducer. The complex was purified using a 1-ml HiTrap-chelating Ni2+ column (GE Healthcare) with a non-linear imidazole gradient in Tris-HCl, 50 mm, pH 8, NaCl, 100 mm, MgCl2, 1 mm, ADP, 0.5 mm, and glycerol 10% (v/v). Imidazole concentrations used were 10, 50, 100, 300, and 500 mm; the HisDraT-GlnB complex eluted at 300 mm. After elution with imidazole from the HiTrap chelating Ni2+ column, HisDraG, HisDraT, and the DraT-GlnB complex were partially precipitated. The samples were centrifuged at 10,000 × g for 10 min at 4 °C, and only the supernatant was used for further analysis. Proteins were dialyzed to remove imidazole. Typical protein preparations were more that 90% homogeneous judged by densitometry analysis of Coomassie Blue-stained SDS-PAGE gel. Proteins were kept at –80 °C until use. In Vitro Uridylylation of GlnB and GlnZ-In vitro uridylylation of GlnB and GlnZ was performed as described previously (30Araujo L.M. Huergo L.F. Invitti A.L. Gimenes C.I. Bonatto A.C. Monteiro R.A. Souza E.M. Pedrosa F.O. Chubatsu L.S. Braz. J. Med. Biol. Res. 2008; 41: 289-294Crossref PubMed Scopus (22) Google Scholar). The products of the reactions were analyzed by native gel electrophoresis and MALDI-TOF spectrometry as described (30Araujo L.M. Huergo L.F. Invitti A.L. Gimenes C.I. Bonatto A.C. Monteiro R.A. Souza E.M. Pedrosa F.O. Chubatsu L.S. Braz. J. Med. Biol. Res. 2008; 41: 289-294Crossref PubMed Scopus (22) Google Scholar). After the uridylylation reaction, His-GlnD was removed from the system using MagneHis-Ni2+ beads (Promega Co, Madison, WI). Fully uridylylated GlnB and GlnZ were dialyzed against 50 mm Tris-HCl, pH 7.5, 0.1 m KCl, 20% glycerol overnight at 4 °C to remove ATP and 2-OG. Pull-down Assays-In vitro complex formation was performed using MagneHis-Ni2+ beads according to the manufacturer’s instructions (Promega). For HisDraG-GlnZ interaction the reactions were conducted in buffer containing 50 mm Tris-HCl, pH 8, 0.1 m NaCl, 0.05% (w/v) LDAO, 10% glycerol, 20 mm imidazole, and 1 mm MgCl2 in the presence or absence of effectors (2-OG, ATP, ADP, AMP) as indicated in each experiment. For HisDraT-GlnB interaction, the same buffer was used except that LDAO was substituted by Tween 20 (0.05%, v/v). Ten microliters of beads were equilibrated by two washes with 100 μl of buffer. Binding reactions were performed in 500 μl of buffer by adding purified proteins to 0.5 μm. Protein concentrations were calculated assuming GlnB and GlnZ to be trimers and DraG and DraT to be monomers. After 5 min of incubation at room temperature, the beads were washed three times with 250 μl of buffer. Elution was performed by incubating the beads either with 20 μl of buffer containing 0.5 m imidazole for 5 min (when His-DraG was used as bait) or with 30 μl of SDS-PAGE sample buffer 1× and boiled for 5 min (when His-DraT was used as bait). Eluted samples were analyzed in 12.5% SDS-PAGE stained with Coomassie Blue. For complex dissociation assays, the beads were washed with 20 μl of buffer containing the effectors prior to elution. Quantitative SDS-PAGE Analysis of the DraT-GlnB Complex-Samples were subjected to SDS-PAGE 12.5% and stained with SYPRO-Ruby (Invitrogen). The intensity of the bands was converted to protein concentration using a calibration curve of both pure His-DraT and GlnB present in the same gel. Each gel was loaded with five different dilutions of the standards GlnB (1.69, 3.37, 6.74, 13.47, and 26.94 pmol of trimer), His-DraT (1.68, 3.35, 6.69, 13.39, and 26.78 pmol of monomer) and the pure DraT-GlnB complex (4, 2, 1, 0.5, and 0.25 μg). The amount of protein loaded was determined by the Bradford assay. The SYPRO-Ruby signal was recorded using a Biochemi system (UVP) and quantified using the ImageQuant software (GE Healthcare). Gel Filtration-Gel filtration chromatography was performed on a Superose 12 10/30 GL column using 50 mm Tris-HCl, pH 8.0, 10% (v/v) glycerol, 0.05% (w/v) Tween 20, 100 mm NaCl, 1 mm MgCl2, 1 mm ADP as buffer. The elution volumes indicated are an average of two independent runs. The column was calibrated with molecular mass markers from Sigma (α-amylase, alcohol dehydrogenase, bovine serum albumin, carbonic anhydrase, and cytochrome c). Purified proteins (HisDraT and/or GlnB) were incubated in 50 mm Tris-HCl, pH 8.0, 10% (v/v) glycerol, 100 mm NaCl, 1 mm MgCl2, and 1 mm ADP for 20 min on ice before loading. The loaded samples consisted of GlnB (4 μmol as trimer), HisDraT (4 μmol as monomer), or HisDraT plus GlnB (4 μmol each) in 400 μl. Amino Acid Composition-The amino acid composition of the purified DraT-GlnB complex was determined as described (34Durand M. Merrick M. J. Biol. Chem. 2006; 281: 29558-29567Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). To calculate the DraT:GlnB stoichiometry, we used a mathematical model in which we compared the values of the experimental amino acid composition of the complex with the respective amino acid compositions of DraT and GlnB. Asparagine and glutamine are, respectively, converted to aspartate and glutamate; therefore, Asx and Glx correspond to the amount of Asp + Asn and Glu + Gln, because Asn and Gln are deamidated following acid treatment. Norleucine was used as an internal standard, but is not reported. We used the data from two different estimations of amino acid composition to calculate the stoichiometry (s) of the DraT-GlnB complex, with s representing the number of GlnB mol for 1 mol of DraT (DraT:s GlnB). The value of s was calculated using the Solver tool in Microsoft Excel to minimize the mean square deviation between the calculated fractions of each amino acid in the complex and the fractions obtained with the experimental data. The calculated fraction was obtained using Equation 1,naaDraT-GlnBNDraT-GlnB=naaDraT+(s×naaGlnBNDraT+(s×NGlnB)Eq. 1 where naa is the number of one particular amino acid in DraT, GlnB, or DraT-GlnB and N is the number of all the amino acids used for the calculation in DraT, GlnB, or DraT-GlnB. All the amino acids reported in supplemental Table S1 were included in the calculation. Valine and isoleucine data were not used, because upon acidic hydrolysis the peptide bonds of Ile-Ile, Val-Val, Ile-Val, and Val-Ile are only partially cleaved, and consequently the values obtained for Val and Ile can be underestimated. DraT and GlnB do not have comparable numbers of those peptide bonds, and, therefore, we did not consider those two residues for estimation of the stoichiometry. DraG Interaction with GlnZ and GlnZ-UMP3 in Vitro-We have previously shown that DraG can interact with both uridylylated and de-uridylylated GlnZ in vivo and that de-uridylylated GlnZ-DraG interaction is stabilized by ADP. The binary complex contains a 1:1 ratio of GlnZ trimer/DraG monomer (10Huergo L.F. Chubatsu L.S. Souza E.M. Pedrosa F.O. Steffens M.B. Merrick M. FEBS Lett. 2006; 580: 5232-5236Crossref PubMed Scopus (36) Google Scholar, 12Huergo L.F. Merrick M. Pedrosa F.O. Chubatsu L.S. Araujo L.M. Souza E.M. Mol. Microbiol. 2007; 66: 1523-1535PubMed Google Scholar). To characterize further the effects of GlnZ uridylylation on interaction with DraG, we purified GlnZ and derived GlnZ-UMP3 by in vitro uridylylation (supplemental Fig. S1). In vitro complex formation between HisDraG and either GlnZ or GlnZ-UMP3 was assayed by pull-down using MagneHis-Ni2+ beads in the presence of ATP, ADP, and 2-OG. Control experiments indicated that neither GlnZ nor GlnZ-UMP3 bind to the Ni2+ beads under the conditions used (data not shown). Thus, the presence of GlnZ in the eluate indicates interaction with HisDraG. Both GlnZ and GlnZ-UMP3 co-purified with HisDraG in the presence of ADP but not in the presence of ATP (Fig. 1) or AMP (data not shown). 2-OG negatively influenced complex formation in the presence of ADP. Fig. 1 shows that the effects of ATP, ADP and 2-OG were not significantly changed whether GlnZ was uridylylated or not (compare Fig. 1, A and B). To determine which form of GlnZ binds preferentially to HisDraG we challenged HisDraG with equimolar amounts of GlnZ and GlnZ-UMP3 in the presence of ADP. The equimolar GlnZ:GlnZ-UMP ratio was confirmed by analysis on SDS-PAGE (Fig. 2A, lane 1 and supplemental Fig. S2). Quantification of GlnZ after elution of the pull-down assay using HisDraG as bait showed a GlnZ:GlnZ-UMP ratio of ∼4 (Fig. 2A, lane 2 and supplemental Fig. S2), a result reproduced in two independent experiments. These results, which are in good quantitative agreement with our previous in vivo data (10Huergo L.F. Chubatsu L.S. Souza E.M. Pedrosa F.O. Steffens M.B. Merrick M. FEBS Lett. 2006; 580: 5232-5236Crossref PubMed Scopus (36) Google Scholar), confirm that DraG can bind to both GlnZ or GlnZ-UMP3 in the presence of ADP but that the interaction is about four times more favorable when GlnZ is de-uridylylated.FIGURE 2Competition assay between GlnZ and GlnZ-UMP3 to bind DraG. A, complex formation was assessed by co-precipitation using Ni2+ beads. Reactions were performed in the presence of ADP. Binding reactions were conducted in 500 μl of buffer, adding the purified proteins at concentrations of 0.5 μm HisDraG and 0.5 μm GlnZ plus GlnZ-UMP3. The eluted fraction (2Nordlund S. Triplett E.W. Prokaryotic Nitrogen Fixation: A Model System for Analysis of Biological Processes. Horizon Scientific Press, Wymondham, UK2000: 149-167Google Scholar) and an equimolar mixture of GlnZ and GlnZ-UMP3 (1Dixon R. Kahn D. Nat. Rev. Microbiol. 2004; 2: 621-631Crossref PubMed Scopus (729) Google Scholar) were subjected to SDS-PAGE and the gel was Coomassie Blue-stained. Arrows indicate the identified proteins. MW indicates molecular weight markers in kDa. B, native-PAGE of GlnZ (1Dixon R. Kahn D. Nat. Rev. Microbiol. 2004; 2: 621-631Crossref PubMed Scopus (729) Google Scholar), GlnZ-UMP3 (2Nordlund S. Triplett E.W. Prokaryotic Nitrogen Fixation: A Model System for Analysis of Biological Processes. Horizon Scientific Press, Wymondham, UK2000: 149-167Google Scholar), and an equimolar mixture of GlnZ and GlnZ-UMP3 (3Zhang Y. Burris R.H. Ludden P.W. Roberts G.P. FEMS Microbiol. Lett. 1997; 152: 195-204Crossref PubMed Google Scholar). The gels were Coomassie Blue-stained.View Large Image Figure ViewerDownload Hi-res image Download (PPT) It has been reported that PII proteins from some organisms can form heterotrimers when mixed in solution (35Forchhammer K. Hedler A. Strobel H. Weiss V. Mol. Microbiol. 1999; 33: 338-349Crossref PubMed Scopus (45) Google Scholar). To confirm that the results reported in Fig. 2A were not caused by the formation of heteromers, i.e. GlnZ-UMP1 or GlnZ-UMP2, we mixed equimolar amounts of GlnZ and GlnZ-UMP3, kept these proteins at room temperature for 20 min and then assessed the quaternary structure by native-PAGE. The results in Fig. 2B show that there was no detectable heterotrimer formation under the conditions tested (compare with supplemental Fig. S1B). We did not detect co-purification of either GlnB or GlnB-UMP3 with HisDraG using ADP in buffers (data not shown). This suggests that DraG cannot interact with GlnB and confirms our previous in vivo data (10Huergo L.F. Chubatsu L.S. Souza E.M. Pedrosa F.O. Steffens M.B. Merrick M. FEBS Lett. 2006; 580: 5232-5236Crossref PubMed Scopus (36) Google Scholar). DraT Interaction with GlnB and GlnB-UMP3 in Vitro-We have previously shown that DraT can interact with de-uridylylated GlnB in vivo (10Huergo L.F. Chubatsu L.S. Souza E.M. Pedrosa F.O. Steffens M.B. Merrick M. FEBS Lett. 2006; 580: 5232-5236Crossref PubMed Scopus (36) Google Scholar). To characterize the influence of GlnB effectors (ATP, ADP, AMP, and 2-OG) and GlnB uridylylation status on the interaction with DraT we performed pull-down assays where HisDraT was immobilized on nickel beads and challenged with either GlnB or GlnB-UMP3. Negative controls indicate that neither GlnB nor GlnB-UMP3 bind to the beads under the conditions used (data not shown). GlnB co-purified with HisDraT in all conditions tested although there were substantial differences in the amount of GlnB recovered with HisDraT according to the presence of nucleotides and/or 2-OG in the buffers (Fig. 3A and supplemental Fig. S3A). The GlnB signal in the eluate was stronger when ADP was present (Fig. 3A; lanes 7, 8, and 9) and addition of 2-OG did not alter the capacity of GlnB to interact with HisDraT. However, when ATP was used alone the GlnB signal in the eluate decreased 2-fold when compared with ADP (compare Fig. 3A, lanes 4 and 7). The combination of ATP and 2-OG almost abolished complex formation (Fig. 3A, lanes 5 and 6). AMP was also capable of stimulating complex formation (Fig. 3A, compare lanes 1 and 10) and the AMP-stimulating effect was slightly counteracted by the presence of 2-OG (Fig. 3A, compare lanes 10 and 12). When the experiment in Fig. 3A was performed in the presence of the DraT substrate, NAD+ 1 mm, similar results were obtained (data not shown). To confirm the negative effects of ATP in combination with 2-OG on HisDraT-GlnB complex formation we immobilized the complex on Ni2+ beads in the presence of ADP and then subjected the sample to a wash step in buffer containing ADP, ADP plus 2-OG, ATP, or ATP plus 2-OG. The results (Fig. 4) confirmed that the DraT-GlnB complex is destabilized in the presence of a combination of ATP and 2-OG and that dissociation of the complex is further enhanced by a high concentration of 2-OG. We also evaluated the effects of different combinations of ADP and ATP on HisDraT-GlnB complex formation, under low (0.1 mm) or high (2 mm) concentrations of 2-OG. ATP inhibited complex formation in a 2-OG concentration-dependent manner despite the presence of ADP in the buffers (Fig. S4), indicating that ATP and ADP compete for the nucleotide-binding sites of GlnB. Unexpectedly, when we challenged immobilized HisDraT to bind GlnB-UMP3 we observed a different response in comparison to results obtained with de-uridylylated GlnB (compare Fig. 3, A and B and supplemental Fig. S3, A and B). We did not detect interactions between HisDraT and GlnB-UMP3 in the absence of nucleotides or in the presence of AMP (Fig. 3B, lanes 1–3 and 10–12). A considerably different pattern of complex formation in response to ATP was observed depending on whether GlnB was uridylylated or not (compare Fig. 3, A and B, lanes 4–6). ATP-stimulated complex formation with de-uridylylated GlnB but not with GlnB-UMP3. Furthermor" @default.
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• W2051200740 title "In Vitro Interactions between the PII Proteins and the Nitrogenase Regulatory Enzymes Dinitrogenase Reductase ADP-ribosyltransferase (DraT) and Dinitrogenase Reductase-activating Glycohydrolase (DraG) in Azospirillum brasilense" @default.
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