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Originally published In Press as doi:10.1074/jbc.M301992200 on May 20, 2003

J. Biol. Chem., Vol. 278, Issue 31, 28711-28718, August 1, 2003
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The Amino-terminal GAF Domain of Azotobacter vinelandii NifA Binds 2-Oxoglutarate to Resist Inhibition by NifL under Nitrogen-limiting Conditions*

Richard Little and Ray Dixon {ddagger}

From the Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, United Kingdom

Received for publication, February 25, 2003 , and in revised form, May 6, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The expression of genes required for the synthesis of molybdenum nitrogenase in Azotobacter vinelandii is controlled by the NifL-NifA transcriptional regulatory complex in response to nitrogen, carbon, and redox status. Activation of nif gene expression by the transcriptional activator NifA is inhibited by direct protein-protein interaction with NifL under conditions unfavorable for nitrogen fixation. We have recently shown that the NifL-NifA system responds directly to physiological concentrations of 2-oxoglutarate, resulting in relief of NifA activity from inhibition by NifL under conditions when fixed nitrogen is limiting. The inhibitory activity of NifL is restored under conditions of excess combined nitrogen through the binding of the signal transduction protein Av GlnK to the carboxyl-terminal domain of NifL. The amino-terminal domain of NifA comprises a GAF domain implicated in the regulatory response to NifL. A truncated form of NifA lacking this domain is not responsive to 2-oxoglutarate in the presence of NifL, suggesting that the GAF domain is required for the response to this ligand. Using isothermal titration calorimetry, we demonstrate stoichiometric binding of 2-oxoglutarate to both the isolated GAF domain and the full-length A. vinelandii NifA protein with a dissociation constant of ~60 µM. Limited proteolysis experiments indicate that the binding of 2-oxoglutarate increases the susceptibility of the GAF domain to trypsin digestion and also prevents NifL from protecting these cleavage sites. However, protection by NifL is restored when the non-modified (non-uridylylated) form of Av GlnK is also present. Our results suggest that the binding of 2-oxoglutarate to the GAF domain of NifA may induce a conformational change that prevents inhibition by NifL under conditions when fixed nitrogen is limiting.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The NifL-NifA1 regulatory system controls transcription of nitrogen fixation (nif) genes in response to the redox, nitrogen, and carbon status in Azotobacter vinelandii. The NifL regulatory protein integrates the response to these environmental inputs to regulate the activity of its partner protein, the {sigma}54-dependent transcriptional activator NifA, through protein-protein interactions (1). NifL is a flavoprotein containing FAD as prosthetic group within an amino-terminal PAS domain and is competent to antagonize NifA activity when the flavin moiety is oxidized, irrespective of the nitrogen status (2, 3). The carboxyl terminus of A. vinelandii NifL bears significant homology to the GHKL superfamily of ATPases and histidine protein kinases (4). Although nucleotide hydrolysis or phosphotransfer has not been observed, binding of adenosine nucleotides to the GHKL domain plays a key role in transducing environmental signals by enhancing the inhibitory activity of NifL and stabilizing protein-protein interactions between NifL and NifA (5–7). NifA activity is inhibited by NifL via a concerted mechanism in which catalytic activity, DNA binding, and potentially interaction with RNA polymerase are controlled (8).

NifA is a multidomain protein consisting of a carboxyl-terminal DNA-binding domain, a central catalytic (AAA+) domain required to couple nucleotide hydrolysis to activation of the {sigma}54-RNA polymerase holoenzyme, and an amino-terminal domain of unknown function (9, 10). The amino terminus of NifA comprises a GAF domain, a ubiquitous signaling motif found in signaling and sensory proteins from all three kingdoms of life (11, 12). GAF and PAS domains share strong structural similarities, suggesting an evolutionary relationship, and in common with the PAS domain family, GAF domains are able to bind small molecule co-factors such as 3',5' cyclic guanosine monophosphate and formate (13–15). This raises the possibility that the binding of a co-effector to NifA may be an integral part of nif gene regulation.

We have recently demonstrated that the NifL-NifA system responds in vitro to the small metabolite 2-oxoglutarate, which counteracts the inhibitory function of NifL that is activated by the binding of adenosine nucleotides (16). The response to 2-oxoglutarate in vitro is within the physiological range observed in Escherichia coli, which varies from ~100 µM under conditions of nitrogen excess to ~1 mM under conditions of nitrogen limitation (17). When expressed in E. coli, the A. vinelandii NifL-NifA system is also apparently responsive to the 2-oxoglutarate concentration in vivo (18). 2-Oxoglutarate is an intermediate in the tricarboxylic acid cycle and provides the carbon skeleton for nitrogen assimilation; hence 2-oxoglutarate signals the carbon status (19). This metabolite is also an allosteric effector of the PII-like signal transduction proteins, which mediate nitrogen metabolism and regulation, dependent upon their uridylylation status (19, 20). Under nitrogen excess conditions, the A. vinelandii PII protein (Av GlnK) is primarily in its unmodified form and interacts with the carboxyl-terminal kinase-like domain of NifL to promote inhibition of NifA (21, 22). However, this interaction is relatively insensitive to the concentration of 2-oxoglutarate, unlike the interactions between enteric PII proteins with their receptors (23, 24). In addition, the interaction between Av GlnK and NifL overrides the effect of 2-oxoglutarate in relieving inhibition of NifA activity, and hence NifL is restored to its inhibitory form (16). Under conditions of fixed nitrogen limitation, Av GlnK is uridylylated, and the fully modified form of the protein, GlnK-UMP, is not competent to interact with NifL, allowing the NifL-NifA system to respond to 2-oxoglutarate (16, 21). We therefore propose that the activity of the nitrogen fixation-specific regulatory proteins is responsive to 2-oxoglutarate under nitrogen-limiting conditions (16).

In this report we have used purified components to investigate the interaction of 2-oxoglutarate with NifA and/or NifL. We observe specific interaction of 2-oxoglutarate with the NifA protein, and we demonstrate that this binding is a property of the isolated GAF domain of NifA. We further demonstrate that a truncated form of NifA, NifA-(191–522), lacking the GAF domain, is unable to bind 2-oxoglutarate, and although this truncated protein is inhibited by NifL, it is not responsive to 2-oxoglutarate. Furthermore, we show that the presence of 2-oxoglutarate alters the protease sensitivity of the GAF domain of NifA, suggesting that binding of this ligand may bring about a conformational change in the protein, enabling it to resist inhibition by NifL.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids for Overexpression—Plasmids pTJ40, pTJ54, pDB737, and pAS1, used for overexpression of NifLHis6, NifL-(147–519)His6, NifA, and NifA-(191–522), respectively, have been described previously (7, 8). Plasmid pTM17 expressing the GAF domain of A. vinelandii NifA with a carboxyl-terminal hexahistidine tag (Av NifA-(1–203)His6) was prepared using primers "nifa-3" (5'-ATGTTCTCGAAGCCGTACTCGAGGCGCACCTCGCG-3') and "05-97" (5'-GGGAATGCATATGAATGCAACCATCC-3') to amplify a fragment containing residues 1–203 with plasmid pDB737 as a template; this resulted in the introduction of a new XhoI site downstream of the coding sequence for residue 203. Following NdeI/XhoI digestion, this fragment was cloned into pET23(b) vector (Novagen). Plasmid pRL08, which expresses the 180 amino-terminal residues of A. vinelandii NifA with an amino-terminal hexahistidine tag, Av NifAHis6-(1–180), was prepared by amplification with primers "Av GAFFor" (5'-GCAGCGGCCTGGTGCCGCGCGGCAGCC-3') and "Av180 Rev" (5'-CGATGGATCCTTACAGGCGCACGGTCTGCGCCAGCAGG-3') using pMB737, which expresses NifA in pET28b(+), as a template. After digestion with NdeI and BamHI, the fragment was cloned into pET15b. Plasmid pRL09, which expresses the first 178 residues of Klebsiella pneumoniae NifA with an amino-terminal hexahistidine tag, Kp NifAHis6-(1–178), was prepared by amplification and cloning into pET15b as described for pRL08, using primers "Kp180For" (5'-GGCATTCCTCATATGATCCATAAATCCGATTCGGACACCACCG-3') and "Kp180Rev" (5'-CAACCGGATCCTTACAGGCGAATCGTCTGGGCGATCAG-3'). Nucleotide sequences of the PCR-generated DNA were found to be identical to those in sequence data bases.

Protein Purification—In all cases protein overexpression was achieved in E. coli strain BL21 (DE3) pLysS (Novagen). Cultures were grown aerobically in Luria-Bertani medium, and expression from the T7 promoter was induced by addition of isopropyl-{beta}-D-thioglactopyranoside to 1 mM. A. vinelandii NifA, NifA-(191–522), Av GlnK, K. pneumoniae {sigma}54, and IHF were purified as described previously (8, 16). NifL-(147–519)His6, Av NifA-(1–203)His6, Av NifAHis6-(1–180), and Kp NifAHis6-(1–178) were purified by metal chelate affinity chromatography. Two 1-ml HiTrap chelating HP columns (Amersham Biosciences) were connected in series and equilibrated with buffer A (50 mM potassium phosphate, 200 mM NaCl, 20 mM imidazole, pH 8.0). Purification was carried out using Biocad Sprint perfusion chromatography (PE Biosystems). Following immobilization of the target proteins, the columns were further washed with buffer A containing 10% glycerol. Protein was eluted using a linear gradient from 0 to 500 mM imidazole over a 30-ml elution volume.

Open Complex Assays—NifA-promoted catalysis of open promoter complex formation by {sigma}54-RNA polymerase was used to assay NifA activity and its inhibition by NifL as described previously (16, 21). Linearized template DNA was provided by digesting plasmid pNH8 with EcoRI and BamHI to yield a 260-bp fragment, which contained the K. pneumoniae nifH promoter and upstream activator sequences and was 3'-end-labeled with [{alpha}-32P]dGTP at the BamHI site. Reactions (final volume of 15 µl) were carried out in TAP buffer (50 mM Tris acetate, 100 mM potassium acetate, 8 mM magnesium acetate, 3.5% polyethylene glycol 8000, 1 mM dithiothreitol, pH 7.9) and contained 5 nM template DNA, 3.4 µg/ml denatured salmon sperm DNA, 125 nM core RNA polymerase, 220 nM {sigma}54, 75 nM IHF, and 3.5 mM ATP. GTP was present at a final concentration of 500 µM prior to heparin challenge to allow formation of initiated complexes, which are more stable than open promoter complexes. An ATP-regenerating system was provided by adding creatine kinase (20 units/ml) and creatine phosphate (12 mM). The reaction components were preincubated for 2 min at 30 °C. NifA alone (control samples) or NifA and NifL were incubated in the presence or absence (control samples) of 2-oxoglutarate for 10 min at 30 °C, and reactions were initiated by the addition of these samples to the reaction components. Following 20 min of incubation, reactions were mixed with 3 µl of dye mix containing 50% glycerol, 0.05% bromphenol blue, 0.1% xylene cyanol, and 2 µg of heparin and immediately loaded onto a 4% (w/v) polyacrylamide gel (acrylamide:bisacrylamide ratio, 80:1) in 25 mM Tris-HCl, 400 mM glycine, pH 8.6, which had been prerun at 180 V at room temperature down to a constant power of 2 W. Gels were run for 2.5–3.0 h at room temperature at 100 V. Gels were dried down, and the percentage of radioactivity in open complexes was quantitated with the Fujix BAS1000 phosphorimager.

Limited Proteolysis—Limited proteolysis was performed at 20 °C in 50 mm Tris acetate, 100 mm potassium acetate, 8 mM magnesium acetate, 1 mM dithiothreitol, pH 7.0. Final concentrations of NifA as well as other proteins and ligands added are indicated in the figure legends. A trypsin:NifA weight ratio of 1:600 was used. 15-µl samples were removed at the time intervals indicated in the figure legends to tubes containing a 10-fold weight excess of soybean trypsin-chymotrypsin inhibitor. To these samples were added 15 µl of gel loading buffer (125 mM Tris-HCl, 4% sodium dodecyl sulfate, 20% glycerol, 10% {beta}-mercaptoethanol, 0.05% bromphenol blue, pH 8.6). Samples were heated at 100 °C for 4 min prior to electrophoretic separation.

Isothermal Titration Calorimetry (ITC)—Experiments were performed in a VP-ITC isothermal titration calorimeter (MicroCal, Inc.) at 28 °C in a cell volume of 1.35 ml as described previously (21). Buffer conditions were 50 mM Tris acetate, 100 mM potassium acetate, 50 mM KSCN, 10% glycerol, 0.1 mM dithiothreitol, pH 7.5. NifA protein samples were dialyzed overnight at 4 °C prior to ITC, and protein concentrations were determined by the Bradford method using bovine serum albumin as the standard. Ligands were titrated from a 250-µl injection syringe into the sample cell, which was stirred at 450 rpm. Volumes of the ligand injections are indicated in the figure legends. The heat change for the dilution of the ligand in the absence of protein was measured for each experiment and was subtracted from the measured heat change of ligand binding to protein. Data analysis was performed with the Origin program, provided by MicroCal, using equations and curve-fitting analysis to obtain least squares estimates of the binding enthalpy, stoichiometry, and binding constant (25). Binding stoichiometries were derived on the assumption that proteins and ligand were fully active with respect to binding.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The GAF Domain of NifA Is Required for the Response to 2-Oxoglutarate—The role of the amino-terminal GAF domain of NifA proteins is not well defined, although in the gamma proteobacteria, which co-express nifL with nifA, this domain may play a role in modulating the NifL-NifA interaction. The inhibitory activity of NifL is stimulated by adenosine nucleotides to promote formation of the NifL-NifA complex (5–7). This protein-protein interaction sequesters NifA and inhibits ATP hydrolysis by the activator, thus preventing productive interactions with {sigma}54-RNA polymerase and inhibiting transcriptional activation at nif promoters. We have demonstrated previously that the NifL-NifA system is directly responsive to the small molecular weight effector 2-oxoglutarate, which acts allosterically to relieve inhibition by NifL in the presence of adenosine nucleotides (16), thus enabling NifA to interact with {sigma}54-RNA polymerase holoenzyme. Because we had not detected any interaction between 2-oxoglutarate and NifL, we suspected that the amino-terminal GAF domain of NifA may be required for the response to this effector. Accordingly, we examined whether a truncated form of NifA lacking the GAF domain, NifA-(191–522), is responsive to 2-oxoglutarate in the presence of NifL. This truncated protein retains the ability to activate transcription and is responsive to NifL (8). For these assays, we measured the formation of open promoter complexes by NifA in the presence of {sigma}54-RNA polymerase holoenzyme and integration host factor at the nifH promoter (5). Open promoter complex formation by NifA was measured in the presence and absence of a truncated form of NifL, NifL-(147–519)His6, which lacks the redox-responsive PAS domain and thus avoids the necessity to perform assays under reducing conditions (7).

Consistent with previous results, we observed that inhibition of wild-type NifA activity by NifL-(147–519)His6 in the presence of adenosine nucleotides was ablated at physiologically relevant concentrations of 2-oxoglutarate (Fig. 1, open squares). The assay was responsive between 0.01 and 2 mM 2-oxoglutarate to yield almost total relief of NifA activity from inhibition by NifL. However the response to 2-oxoglutarate was markedly different when the truncated NifA protein, NifA-(191–522), was examined. Although this truncated protein is susceptible to inhibition by NifL-(147–519)His6, little relief of inhibition was observed at concentrations of 2-oxoglutarate across the physiological range (Fig. 1, open triangles). Control experiments with NifA and NifA-(191–522) showed that these proteins did not give a significant response to 2-oxoglutarate in the absence of NifL (Fig. 1). These observations suggest that the GAF domain is necessary for the response to 2-oxoglutarate and that this response is exerted either directly or indirectly through the amino terminus of the NifA protein.



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  		FIG. 1.
Influence of 2-oxoglutarate on the ability of NifL to inhibit wild-type NifA or a truncated version of NifA lacking the GAF domain. NifA activity was measured by the formation of open promoter complexes as described under "Experimental Procedures" and plotted on the y axis relative to the extent of NifA or NifA-(191–522) activity in the absence of 2-oxoglutarate. Reactions contained 200 nM (dimer) NifA and 150 nM (dimer) NifL-(147–519)His6 (squares), 200 nM (dimer) NifA-(191–522) and 400 nM (dimer) NifL-(147–519)His6 (triangles), 200 nM NifA (dimer) alone (circles), or 200 nM (dimer) NifA-(191–522) alone (dotted line). 2-Oxoglutarate concentrations are indicated on the x axis.

 

Stochiometric Binding of 2-Oxoglutarate to NifA Measured by Isothermal Titration Calorimetry—We sought to directly measure binding of 2-oxoglutarate to the NifA protein using isothermal titration calorimetry. Titration of wild-type NifA with 2-oxoglutarate gave a KD of ~57 µM (Fig. 2A, squares). Enthalpy values were negative, demonstrating that the binding of 2-oxoglutarate is an exothermic process. Data analysis of the binding isotherm gave a best fit to a single-site model with a binding stoichiometry of ~1. The dissociation constant for the binding of 2-oxoglutarate to NifA compares favorably with the previously determined apparent kact of ~150 µM for the interaction of 2-oxoglutarate with the NifL-NifA complex in vitro (16). The open complex assays demonstrated previously that this interaction was highly specific to 2-oxoglutarate, as 3-oxoglutarate and 2-oxobutyrate were not competent to relieve inhibition by NifL (16). To confirm the specificity of the interaction, a second portion of the same NifA sample was titrated with 3-oxoglutarate in the concentration range of 0–500 µM. No interaction was detected with 3-oxoglutarate across this concentration range (Fig. 2A, open circles). In comparable ITC experiments, we did not observe an interaction of 2-oxoglutarate with NifLHis6 (data not shown), indicating that this ligand binds to NifA but not to NifL.



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  		FIG. 2.
Calorimetric titration of the binding of 2-oxoglutarate to NifA. A, binding isotherms upon injecting either 2-oxoglutarate (filled squares) or 3-oxoglutarate (open circles) into wild-type NifA (22.6 µM monomer). 5-µl injections of ligand (2.5 mM) were made to the stoichiometric excess given in the figure. The integrated heats of reaction are plotted against the molar ratio of total ligand concentration to total protein concentration. The solid line shows the best fit to the data according to an equivalent site binding model with n = 0.95 ± 0.2 sites/molecule, KD = 57 ± 5.0 µM, and {Delta}H = –3.4 ± 0.9 kcal/mol. B, binding isotherm upon injecting 2-oxoglutarate (filled squares) or 3-oxoglutarate (open circles) into Av NifA-(1–203)His6 (381 µM monomer). 3- or 10-µl injections of ligand (2.5 mM) were made to the stoichiometric excess given in the figure. The solid line shows a best fit to the data according to an equivalent site binding model with n = 0.82 ± 0.01 sites/molecule, KD = 52 ± 0.4 µM, and {Delta}H = –10.6 ± 0.2 kcal/mol.

 

To investigate the possibility that 2-oxoglutarate interacts directly with the amino-terminal GAF domain of NifA, a construct comprising residues 1–203 with a carboxyl-terminal hexahistidine tag was prepared, and the expressed polypeptide was purified by nitrilotriacetic acid affinity chromatography. Titration of Av NifA-(1–203)His6 with 2-oxoglutarate gave a KD of ~52 µM (Fig. 2B, squares). Data analysis of the binding isotherm gave a best fit to a single-site model with a binding stoichiometry of ~0.82. Titration of 3-oxoglutarate with a second portion of the same Av NifA-(1–203)His6 preparation confirmed the specificity of the 2-oxoglutarate interaction (Fig. 2B, circles). Hence, these data show that 2-oxoglutarate, but not 3-oxoglutarate, binds to the isolated GAF domain of NifA. We also tested whether NifA could bind 2-oxoglutarate in the absence of the GAF domain using the truncated NifA derivative, NifA-(191–522). Titration up to a 54-fold excess of 2-oxoglutarate with NifA-(191–522) elicited only a very weak near-linear response (data not shown) with a fit to a KD of ~7 mM, perhaps indicative of nonspecific binding. These results concur with the open complex assays and suggest that the GAF domain is the major site on the A. vinelandii NifA protein for the binding of 2-oxoglutarate.

Sequence alignments with YKG9, the structural prototype for the GAF domain (12), indicate that the GAF domain of A. vinelandii NifA extends to residue 180. To determine whether this "minimal" GAF domain was competent to bind 2-oxoglutarate, we engineered a construct expressing residues 1–180. This polypeptide, Av NifAHis6-(1–180), gave a KD of ~80 µM for binding to 2-oxoglutarate with a binding stoichiometry of ~1 (Fig. 3A). Although GAF domains from other NifA proteins are not highly conserved, it is possible that the binding of 2-oxoglutarate could provide a common mechanism for regulating NifA activity, particularly for those NifA proteins which are inhibited by a NifL-like protein. The well-studied K. pneumoniae NifL and NifA proteins have several characteristics in common with their A. vinelandii counterparts, although these proteins have not been tractable to purification in their native forms (1). To determine whether NifA proteins from both organisms have common ligand binding properties, we expressed and purified the K. pneumoniae NifA GAF domain, which shows 44% identity to that of A. vinelandii. However, in contrast to the A. vinelandii GAF domain, binding of 2-oxoglutarate to the K. pneumoniae GAF domain construct, Kp NifAHis6-(1–178), was not detected by ITC (Fig. 3B).



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  		FIG. 3.
Isothermal titration profiles comparing the interaction of 2-oxoglutarate with the GAF domains of A. vinelandii and K. pneumoniae. The upper panels show the raw data for the heat effect during titration of 2-oxoglutarate, and the lower panels show the binding isotherms. The protein concentration in each case was 33 µM (monomer), and injections of 2-oxoglutarate (2 mM) were either 2, 5, or 10 µl. A, binding to Av NifAHis6-(1–180). The best fit to the data gave n = 1.09 ± 0.15 and KD = 79.6 ± 6.4 µM. B, data for Kp NifAHis6-(1–178). The data could not be fitted easily to any model and indicated a KD in excess of 5 mM.

 

The Interaction of 2-Oxoglutarate with NifA Enhances Proteolytic Cleavage of the GAF Domain—We have shown previously that the amino-terminal region of NifA is particularly susceptible to proteolytic cleavage by trypsin. In the presence of nucleotides such as MgADP or MgATP{gamma}S, which bind to the central domain, the major cleavage products are 40- and 35-kDa polypeptides (which represent the central plus the carboxyl-terminal domain and the isolated central domain, respectively) and a 20-kDa polypeptide designated A6, which is a central domain subdomain fragment (Fig. 4A) (26). The amino termini of these polypeptides result from cleavage at Arg-202, located within the linker between the GAF and central domains. We have also detected cleavages within the GAF domain itself at Arg-8, Arg-70, and Arg-165 (26). We performed similar experiments to determine whether 2-oxoglutarate influences the pattern of proteolytic digestion of the GAF domain. Control experiments with wild-type NifA in the presence of 3.5 mM ATP revealed a similar pattern of digestion to that observed previously (Fig. 4B). The GAF domain was cleaved to yield the A1–A3 polypeptides, and we detected cleavage at Arg-202 to yield the 40-kDa central and DNA-binding domain fragment (A4) as well as the 35-kDa central domain polypeptide (A5) and its subfragment (A6). When NifA was preincubated with 2 mM 2-oxoglutarate, a similar pattern of digestion was observed, but the amino terminus was degraded far more rapidly to yield the A4, A5, and A6 polypeptides (Fig. 4B, lanes marked +). This suggests that 2-oxoglutarate exposes the amino terminus to cleavage by trypsin, probably as a consequence of a conformational change induced upon binding of this ligand. The change in trypsin sensitivity is apparently specific to 2-oxoglutarate, as the pattern of digestion was indistinguishable from that in the absence of ligand when 3-oxoglutarate was present (Fig. 4B, lanes marked 3).



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  		FIG. 4.
Influence of 2-oxoglutarate on limited trypsin proteolysis of wild-type NifA. A, map of NifA tryptic peptides identified previously from limited proteolysis studies (26). The amino-terminal GAF domain of NifA is shown as open boxes, the central AAA+ domain in gray, and the DNA-binding domain with the HTH motif in black. B, influence of 2-oxoglutarate on the digestion pattern. All reactions contained NifA (6 µM dimer) and ATP (3.5 mM). Samples were incubated in the absence of an additional ligand (lanes marked with a – sign above) or contained 2 mM 2-oxoglutarate (lanes marked with a + sign above) or 2 mM 3-oxoglutarate (lanes marked with a 3 above). Lanes 3, 6, 9, 12, 15, 18, and 21 contained NifA and ATP incubated for t = 0, 2, 6, 10, 30, 60, and 100 min, respectively. Lanes 4, 7, 10, 13, 16, 19, and 22 contained NifA, ATP, and 2-oxoglutarate incubated for t = 0, 2, 6, 10, 30, 60, and 100 min, respectively. Lanes 5, 8, 11, 14, 17, 20, and 23 contained NifA, ATP, and 3-oxoglutarate incubated for t = 0, 2, 6, 10, 30, 60, and 100 min, respectively. Lane 2 shows purified NifA as a marker. Lanes 1 and 24 show markers with molecular masses in kilodaltons.

 

We have shown previously that when the NifL-NifA complex is formed, NifA is protected from proteolysis at Arg-202 and within the carboxyl-terminal region of the GAF domain, suggesting that NifL may restrict access to these cleavage sites or induce a conformational change (26). Since the binding of 2-oxoglutarate to NifA prevents inhibition by NifL and potentially inhibits the interaction between the two proteins, we re-examined the influence of NifL on proteolysis of NifA in the presence of 2-oxoglutarate. Under the conditions used for proteolysis, NifL remained substantially resistant to trypsin digestion (Fig. 5A, lanes 2–4), and as anticipated, digestion of NifL was not influenced by 2-oxoglutarate (Fig. 5A, compare lanes 11–13 and 14–16). As observed previously in the presence of MgADP, the addition of NifL decreased the rate of trypsin digestion of full-length NifA (26). However, when 2-oxoglutarate was also present, this protection by NifL was significantly diminished (Fig. 5A, compare lanes 11–13 with 14–16), and digestion of NifA was more rapid (Fig. 5B, compare inverted triangles with diamonds). Loss of protection by NifL suggests that 2-oxoglutarate may inhibit the interaction between NifL and NifA, consistent with the increase in NifA activity under these conditions (Fig. 1). However, this relief from inhibition by NifL is overridden under conditions of excess fixed nitrogen. Our previous results demonstrated that the interaction of the non-modified form of Av GlnK with NifL restores inhibition of NifA activity in the presence of 2-oxoglutarate (16, 21). Accordingly, when Av GlnK was also present, the rate of digestion of NifA decreased, implying that NifL again protected sites in NifA from proteolytic cleavage (Fig. 5A, compare lanes 14–16 with 17–19, and Fig. 5B, compare diamonds with circles). A mutant form of Av GlnK, GlnK E44C, which does not interact with NifL (21), did not protect NifA from cleavage in the presence of NifL (data not shown). Control experiments, where NifL was omitted from the reaction, indicated that Av GlnK itself did not protect NifA from digestion (Fig. 5A, lanes 5–10, and Fig. 5B, triangles and squares).



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  		FIG. 5.
Influence of NifL and Av GlnK on limited proteolysis of NifA in the presence of 2-oxoglutarate. A, examples of the original data. All reactions contained 3.5 mM ADP. NifA (5 µM dimer), NifLHis6 (2.5 µM tetramer), Av GlnK (10 µM trimer), and 2-oxoglutarate (4 mM) were added as indicated below. Samples were taken at t = 0 (lanes 2, 5, 8, 11, 14, and 17), t = 6 (lanes 3, 6, 9, 12, 15, and 18), and t = 30 (lanes 4, 7, 10, 13, 16, and 19). Samples contained NifLHis6 (lanes 2–4), NifA plus 2-oxoglutarate (lanes 5–7), NifA plus 2-oxoglutarate plus Av GlnK (lanes 8–10), NifLHis6 plus NifA (lanes 11–13), NifLHis6 plus NifA plus 2-oxoglutarate (lanes 14–16), and NifLHis6 plus NifA plus 2-oxoglutarate plus Av GlnK (lanes 17–19). Lane 1 shows molecular mass markers in kilodaltons. B, quantitative analysis of NifA digestion. The percentage of full-length NifA remaining undigested at the indicated times was quantified using the MacBas Version 2.0 image analysis software (Fuji Photo Film Company Ltd). Reactions contained NifA plus 2-oxoglutarate (triangles), NifA plus 2-oxoglutarate plus Av GlnK (squares), NifLHis6 plus NifA (inverted triangles), NifLHis6 plus NifA plus 2-oxoglutarate (diamonds), and NifLHis6 plus NifA plus 2-oxoglutarate plus Av GlnK (circles).

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Although it is clear that the NifL protein plays a major role in sensing the redox and fixed nitrogen status to control the activity of NifA, our results now demonstrate that its partner, NifA, also has an important role in signal perception through the response to 2-oxoglutarate. This study demonstrates that the GAF domain of A. vinelandii NifA binds one molecule of 2-oxoglutarate per monomer, enabling the protein to resist inhibition by the ADP-bound inhibitory form of NifL (Fig. 6). In the absence of 2-oxoglutarate, the interaction between NifL and NifA inhibits ATP hydrolysis by the activator (Fig. 6A), perhaps as a consequence of intramolecular repression mediated by the GAF domain (8). The limited proteolysis experiments suggest that the interaction with 2-oxoglutarate may alter the conformation of the GAF domain, decreasing the affinity for NifL (Fig. 6B) and hence releasing NifA from the complex to allow nucleotide triphosphate hydrolysis and consequent productive interactions with {sigma}54-RNA polymerase. It is important to note that in the absence of NifL, transcriptional activation by NifA is not influenced by 2-oxoglutarate, and therefore this ligand does not apparently modulate NifA activity per se under our assay conditions (16). A truncated NifA protein lacking the GAF domain exhibits a weaker interaction with NifL but remains responsive to inhibition (6, 8). However, there appears to be an alteration in the response of the truncated protein to NifL because unlike the wild-type protein, the catalytic activity (nucleotide triphosphate hydrolysis) of the central domain is not inhibited by the presence of NifL (8). In this study we have observed that the truncated protein is unable to respond to the presence of 2-oxoglutarate so that NifA activity remains inhibited. We predict that this would lead to a situation in which the truncated NifA is constitutively repressed by NifL in vivo. Unfortunately, we have not been able to test this hypothesis, as truncated forms of NifA lacking the GAF domain appear to be unstable in vivo.2 We have isolated two missense mutations in the NifA GAF domain, L120P and R155C, which render the protein insensitive to inhibition by NifL (27). It is possible that these mutations lock NifA in a conformation that is resistant to NifL, even in the absence of 2-oxoglutarate.



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  		FIG. 6.
Model for regulation of NifA activity in response to NifL, Av GlnK, and 2-oxoglutarate. The PAS and ADP-binding domains of NifL are shown as gray and light gray ovals, respectively. The GAF and AAA+ domains of NifA are depicted in dark gray, and the DNA-binding domain with the HTH motif is shown as a diamond. A, in the presence of ADP, NifL binds to NifA, inhibiting its activity. B, upon binding of 2-oxoglutarate (depicted as a star) to the GAF domain of NifA, NifL is no longer competent to inhibit NifA activity. C, the interaction of the non-modified form of Av GlnK with the carboxyl-terminal domain of NifL restores inhibition even when the 2-oxoglutarate concentration is saturating.

 

The allosteric effect of 2-oxoglutarate on NifA activity in the presence of NifL (Fig. 6B) is apparently overridden under oxidizing conditions or in fixed nitrogen excess. Our biochemical studies have show that the non-modified form of the signal transduction protein Av GlnK, which is prevalent under conditions of fixed nitrogen sufficiency, binds to the carboxylterminal domain of NifL to inactivate NifA (21). This interaction occurs even at high 2-oxoglutarate concentrations in vitro, thus re-establishing inhibition by NifL even when the binding site on the GAF domain of NifA is saturated (Fig. 6C). Hence, the interaction with Av GlnK may induce a conformational change in NifL that compensates for the change brought about by the binding of 2-oxoglutarate to the GAF domain of NifA. The Av GlnK-NifL-NifA interaction is also complicated by the potential involvement of 2-oxoglutarate as an allosteric effector of Av GlnK. However, we have found that the binding constant of Av GlnK for 2-oxoglutarate is relatively high compared with that of enteric PII proteins and that binding of Av GlnK to NifL is not particularly responsive to the 2-oxoglutarate concentration (21).

Our studies have shown that 2-oxoglutarate binds to the GAF domain of A. vinelandii NifA, but not apparently to K. pneumoniae NifA. There are several differences in the mechanism in which the NifL-NifA systems from these organisms respond to fixed nitrogen. In K. pneumoniae, GlnK is required to prevent NifL from inhibiting NifA under nitrogen-limiting conditions (28, 29). Conversely in A. vinelandii, the non-uridylylated form of GlnK is required for NifL to inhibit NifA under conditions of nitrogen excess (21, 22). Under conditions of nitrogen limitation, the binding of 2-oxoglutarate to A. vinelandii NifA is required to prevent inhibition by NifL, whereas in the K. pneumoniae system, the interaction with GlnK appears to provide this function. Therefore, there may not be a requirement for an additional ligand such as 2-oxoglutarate in the K. pneumoniae NifL-NifA system; it is possible that the GAF domain in this case is required for the interaction with GlnK. In diazotrophs that do not contain NifL, such as Azospirillum brasiliense, there is evidence that PII signal transduction proteins directly or indirectly modulate the activity of NifA via the GAF domain (30, 31).

GAF domains are ubiquitous in organisms from all phyla and have been proposed to bind a diverse set of regulatory small molecules (11–13). However, although more than 1200 GAF domain proteins have been identified, ligand binding has only so far been demonstrated with the cyclic nucleotide-responsive GAF domains (12, 15) and the FhlA transcriptional activator, a {sigma}54-dependent activator related to NifA, which is responsive to formate (14, 32, 33). Our observation that NifA binds 2-oxoglutarate thus extends the range of small molecules bound by GAF domains.

The physiological consequences of the interaction between 2-oxoglutarate and NifA are likely to be most apparent under conditions of nitrogen and oxygen limitation. Under nitrogen-limiting conditions, Av GlnK is primarily modified by uridylylation, and Av GlnK-UMP is not competent to interact with NifL (16, 21). Provided that the flavin moiety in the PAS domain of NifL remains in a reduced state and the binding pocket in the carboxyl-terminal domain of NifL is saturated with adenosine nucleotide (7), we predict that the ability of NifL to inhibit NifA under conditions of fixed nitrogen limitation will be dependent upon the concentration of 2-oxoglutarate. Clearly, the NifL-NifA system is responsive to 2-oxoglutarate within the physiological range (17, 18). Although 2-oxoglutarate can undoubtedly provide an indirect readout of the nitrogen status, 2-oxoglutarate provides a key signal of the carbon status (19). Therefore, whereas NifL perceives the oxygen and fixed nitrogen status to control NifA activity, the GAF domain of NifA may provide a sensor for carbon availability via its response to 2-oxoglutarate. The importance of 2-oxoglutarate as a signaling molecule is also beginning to emerge in other systems, particularly the cyanobacteria, whereby the activity of the global nitrogen regulator NtcA is influenced by interaction with 2-oxoglutarate (34–36).


   FOOTNOTES
 
* This work was supported by grants from the Biotechnology and Biological Sciences Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 44-1603-450747; Fax: 44-1603-450778; E-mail: ray.dixon{at}bbsrc.ac.uk.

1 The abbreviations used are: NifL, nitrogen fixation-specific regulatory protein, product of A. vinelandii nifL; NifA, nitrogen fixation-specific regulatory protein, product of A. vinelandii nifA; GAF, cGMP phosphodiesterase, adenylate cyclase, FhlA; IHF, integration host factor; ITC, isothermal titration calorimetry; ATP{gamma}S, adenosine 5'-O-(thio-triphosphate); AAA, ATPases associated with a variety of cellular activities; PAS, Per-Arnt-Sim. Back

2 F. Reyes-Ramirez and R. Dixon, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank Tracy Money and Sara Austin for plasmid pTM17 and Francisca Reyes-Ramirez for performing experiments in vivo with the truncated NifA protein lacking the GAF domain. We also thank Gary Sawers and Mike Merrick for comments on the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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