Ligand Binding Interaction with Nucleotides and 2Oxog Iutarate

A. vinelandii NifL contains a C-terminal HATPase_c domain (Figure 1) which is found in histidine protein kinases and phytochrome-like ATPase. Although NifL does not apparently exhibit either histidine protein kinase or ATPase activity, the C-terminal domain binds adenosine nucleotides (Soderback et al. 1998). The presence of Mg ADP increases the stability of the NifL-NifA complex (Money et al. 1999), leading to inhibition of transcriptional activation by NifA in vitro even when the NifL protein is in its reduced form (Hill et al. 1996). Limited proteolysis experiments suggest that adenosine nucleotide binding promotes conformational changes in the C-terminal domain of NifL (Money et al. 2001; Soderback et al. 1998). Mutagenesis of conserved residues in the HATPase_c domain of NifL which influence nucleotide binding in other members of this family, inactivate the signal transduction function of NifL, so that it is unable to inhibit NifA activity in response to environmental effectors (Perry et al. this volume). Hence nucleotide binding is a major determinant of NifL activity.

We have previously suggested that nucleotide binding by NifL might provide a mechanism for sensing the ATP/ADP ratio, but this seems unlikely, since the binding site would be anticipated to be saturated in vivo. Moreover, the ability of MgADP to override the redox response of NifL in vitro presents a physiological quandary, since NifL would be expected to constitutively inhibit NifA activity, independent of the redox status. However, we have recently observed that the NifL-NifA system is directly responsive to an additional ligand, 2-oxoglutarate, which antagonizes the influence of adenosine nucleotides on NifL activity, thus relieving inhibition by NifL in the presence of ADP (Little et al. 2000). Since 2-oxoglutarate is a key intracellular signal of the carbon status, the response of the NifL-NifA system to this metabolite may reflect an additional physiological switch which deactivates NifL in response to carbon availability. The response of the NifL and NifA proteins to 2-oxoglutarate is within the physiological range and the A. vinelandii NifL-NifA system is apparently responsive to the intracellular level of 2-oxoglutarate in vivo when introduced into E. coli (Reyes-Ramirez et al. 2001).

NifL

NifA

FAD redox sensing

ADP binding domain a54 RNA regulatory Po|ynierase interaction

DNA binding

Figure 1. Domain structure of NifL and NifA. Domains were assigned using SMART (http://SMART.embl-heidelberg.de) with the exception of the DNA binding domain of NifA.

The amino terminal region of A. vinelandii NifA comprises a GAF domain (Figure 1), a ubiquitous motif found in signaling proteins from all kingdoms of life, some of which bind cyclic GMP (Aravind, Ponting 1997). Recent structural determination of Saccharomyces cerevisiae YKG9, a member of the GAF family, reveals that the fold of this motif resembles that of the PAS domain and similarities in the binding pockets of the two motifs have led to the suggestion that the GAF fold may also bind a variety of different co-factors (Ho et al. 2000). The GAF domain of NifA is predicted to have a regulatory function and when this domain is removed, producing a truncated variant of NifA comprising the central and C-terminal domains, the truncated protein is no longer susceptible to inhibition by the oxidized form of NifL in vitro (Barrett et al. 2001). However, the truncated protein is still inhibited by NifL when Mg ADP is present, although co-chromatography experiments suggest that the complex formed under these conditions is less stable than with native NifA (Money et al. 1999). The GAF domain apparently has a role in regulating the nucleoside triphosphatase activity of the central domain of NifA in response to NifL, since the ATPase activity of NifA is no longer inhibited by NifL when the GAF domain is absent (Barrett et al. 2001).

We have observed that the GAF domain is necessary for the response of the NifL-NifA system to 2-oxoglutarate, since when this domain is absent, NifL inhibits NifA activity even at high 2-oxoglutarate concentrations (Figure 2A). Moreover, limited proteolysis experiments suggest that the conformation of NifA alters in the presence of 2-oxoglutarate such that the linker region between the GAF and central domains of NifA is more susceptible to trypsin cleavage (Figure 2B). Since this effect is observed in the absence of NifL, it is likely that 2-oxoglutarate binds to NifA, commensurate with the proposed role of the GAF domain in binding small molecules. The proposed conformational change in NifA elicited by 2-oxoglutarate may thus prevent NifL from inhibiting NifA in the presence of adenosine nucleotides, thus favouring nif gene transcription when both the carbon and nitrogen status are appropriate.

Figure 2. Influence of 2-oxoglutarate on NifA. (A) Role of the GAF domain m the response to 2-oxoglutarate. Inhibition by NifL is relieved m the presence of 2-oxoglutarate with native NifA (squares) but m the absence of the GAF domain no response is observed (triangles). (B) Influence of 2-oxoglutarate on limited digestion of NifA by trypsin. Lane M, molecular weight markers; lane 1, NifA with no addition; lane 2 NifA plus 2-oxoglutarate (2 mM); lane 3, NifA plus 3-oxoglutarate (2 mM). Bands indicated as a, b and c represent previously identified trypsin fragments (Money et al. 2001).

Figure 2. Influence of 2-oxoglutarate on NifA. (A) Role of the GAF domain m the response to 2-oxoglutarate. Inhibition by NifL is relieved m the presence of 2-oxoglutarate with native NifA (squares) but m the absence of the GAF domain no response is observed (triangles). (B) Influence of 2-oxoglutarate on limited digestion of NifA by trypsin. Lane M, molecular weight markers; lane 1, NifA with no addition; lane 2 NifA plus 2-oxoglutarate (2 mM); lane 3, NifA plus 3-oxoglutarate (2 mM). Bands indicated as a, b and c represent previously identified trypsin fragments (Money et al. 2001).

3. Nitrogen Sensing: Av GlnK Interacts with NifL

PH-like signal transduction proteins have been implicated in the regulation of nitrogen fixation in several diazotrophs. The activity of these trimeric proteins is regulated by reversible uridylylation catalyzed by uridylyltransferase (UTase) (Arcondeguy et al. 2001; Ninfa and Atkinson 2000). Under nitrogen-limiting conditions PH-like proteins are uridylylated by the UTase and de-uridylylated under conditions of nitrogen excess. A direct role for PH-like proteins in regulating nitrogen fixation has only recently been established. We have shown that the inhibitory activity of NifL:NifA complex is stimulated by interaction with the non-modified forms of E. coli PII and A. vinelandii GlnK in the presence of adenosine nucleotides (Little et al. 2000). The interaction with PH-like proteins overrides the relief of inhibition observed in the presence of high concentrations of 2-oxoglutarate. We propose that Av GlnK signals the nitrogen status by interaction with the NifL-NifA system under conditions of nitrogen excess and that the inhibitory activity of NifL is relieved by elevated levels of 2-oxoglutarate when Av GlnK is uridylylated under conditions of nitrogen limitation.

GlnK

non-inhibitory

Low 2-ketoglutarate inhibitory

High 2-ketoglutarate

Lred A ^ inhibitory

non-inhibitory

Figure 3. Model for regulation of the NifL-NifA system by Av GlnK m response to the nitrogen status.

In order to elucidate the mechanism of nitrogen sensing by the NifL- NifA system it is necessary to determine which protein component(s) interact with PII-like proteins and to analyze the effectors required for this interaction. Using co-chromatography assays with histidine tagged proteins, we have observed that Av GlnK interacts with NifL in the presence of 2-oxoglutarate and ATP (Figure 4). In contrast, no interaction was observed with NifA (data not shown).

NifL

Figure 4. Co-chromatography of histidine-tagged NifL with Av

GlnK. Final ligand concentrations were: 2-oxoglutarate (2mM);

Lane 3, NifL + Av GlnK + 2-oxoglutarate

Lane 6, NifL + Av GlnK E44C + 2-oxoglutarate + ATP

The role of 2-oxoglutarate in this case probably reflects the requirement for the binding of this ligand to Av GlnK, since no interaction of 2-oxoglutarate was observed with NifL alone in ligand binding assays. As controls for these experiments we have used a mutant form of Av GlnK with a substitution, E44C, in the surface exposed T-loop which has a major role in interaction with receptors. In contrast to wild-type Av GlnK, the purified E44C protein does not stimulate the inhibitory action of NifL in vitro and does not interact with NifL in the co-chromatography assay (Figure 4). Surface plasmon resonance (SPR) experiments using BIAcore instrumentation also indicate that NifL interacts with the Av GlnK protein. In these experiments a histidine-tagged derivative of NifL lacking the redox-responsive PAS domain was coupled to a Ni-NTA chip and Av GlnK was added in the presence of 2-oxoglutarate and Mg ATP. The interaction between non-uridylylated Av GlnK and NifL was Mg and adenosine nucleotide-dependent and was not seen with the Av GlnK E44C mutant, confirming the results of the co-chromatography experiments. Since the interaction was not detectable with a truncated fragment of NifL, which lacks the C-terminal nucleotide binding domain, it apparently requires the C-terminal domain of NifL.

4. Mutations which Influence Signal Transduction

We have isolated a series of mutations in both the GAF domain and the central domain of NifA which prevent inhibition by NifL. Most of these mutations block inhibition by NifL in response to both fixed nitrogen and oxygen, but some mutant NifA proteins can apparently discriminate between the inhibitory forms of NifL present under different environmental conditions. Notably, mutant NifA Y254N shows complete resistance to inhibition by NifL in vivo when cultures are grown anaerobically under conditions of nitrogen excess, but is sensitive to NifL under aerobic growth conditions. The Y254N mutation is located in the central domain of NifA in a predicted a-helix that forms part of the Walker A motif in the AAA+ family of ATPases (Neuwald et al. 1999). Purified NifA Y254N is resistant to inhibition by the ADP bound form of NifL in vitro and is also not responsive when Av GlnK, and 2-ketoglutarate are added to the reaction. However, the mutant protein is susceptible to inhibition by the oxidized form of NifL in the presence of ADP, albeit at higher NifLox concentrations than observed with the wild-type NifA protein. The biochemical results thus concur with the in vivo phenotype of the mutation and indicate that the mutant NifA protein is unable to respond to the nitrogen signal generated via the interaction of NifL with Av GlnK and adenosine nucleotides, but is responsive to the oxidized form of NifL when ADP is present. These results suggest that the nature of the complex formed between reduced NifL and NifA in the presence of Av GlnK is different from that formed between oxidized NifL and NifA. Potentially, different conformers of NifL may be generated in response to discrete signal transduction events. The discrete nature of these events is also notable from the properties of mutations in the central region of NifL which inactivate the redox response but do not influence the response to fixed nitrogen. These mutations presumably do not disable perception of the redox signal per se but interfere with communication of this signal to NifA without apparently affecting transduction of the nitrogen signal (Perry et al. this volume).

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