A flavorubredoxin and its associated oxidoreductase (encoded by norV and norW respectively) detoxify NO (nitric oxide) to form N2O (nitrous oxide) under anaerobic conditions in Escherichia coli. Transcription of the norVW genes is activated in response to NO by the σ54-dependent regulator and dedicated NO sensor, NorR, a member of the bacterial enhancer-binding protein family. In the absence of NO, the catalytic activity of the central ATPase domain of NorR is repressed by the N-terminal regulatory domain that contains a non-haem iron centre. Binding of NO to this centre results in the formation of a mononitrosyl iron species, enabling the activation of ATPase activity. Our studies suggest that the highly conserved GAFTGA loop in the ATPase domain, which engages with the alternative σ factor σ54 to activate transcription, is a target for intramolecular repression by the regulatory domain. Binding of NorR to three conserved enhancer sites upstream of the norVW promoter is essential for transcriptional activation and promotes the formation of a stable higher-order NorR nucleoprotein complex. We propose that enhancer-driven assembly of this oligomeric complex, in which NorR apparently forms a DNA-bound hexamer in the absence of NO, provides a ‘poised’ system for transcriptional activation that can respond rapidly to nitrosative stress.

Introduction

NO (nitric oxide) is an intermediate of respiratory denitrification [1], and is one of the toxic species released by macrophages of the immune system in the defence against invading pathogenic bacteria [2]. As a consequence, bacteria have evolved mechanisms in order to survive nitrosative-induced stress. The Escherichia coli flavorubredoxin and its associated oxidoreductase function under anaerobic conditions to convert NO into N2O (nitrous oxide) and are encoded by the norV and norW genes respectively [3]. Expression of these genes is controlled by norR, which is divergently transcribed upstream of the norVW transcriptional unit. NorR is a bEBP (bacterial enhancer-binding protein) of the AAA+ (ATPase associated with various cellular activities) class of proteins and activates the σ54-dependent transcription of norVW in response to NO [4,5]. NorR and the global regulator, NsrR, are the only known dedicated NO sensors [6].

In contrast to the housekeeping σ70-class of bacterial σ factors, transcription by the σ54-class requires the bEBP to use the energy generated from ATP hydrolysis to drive conformational rearrangements in the σ54-RNA polymerase holoenzyme. As is the case for other AAA+ proteins, bEBPs are competent to hydrolyse ATP when assembled as a hexamer, since the catalytic site is formed by residues from adjacent protomers ([7] and references therein). The central (AAA+) domain (Figure 1A) contains specific structural features that enable nucleotide-dependent interactions with σ54. The highly conserved GAFTGA motif forms part of a surface-exposed loop that contacts σ54 during the ATP hydrolysis cycle [811]. In the ATP-bound transition state, the GAFTGA-containing loop 1, assisted by a second loop (loop 2) is in an extended conformation and is therefore able to establish contact with region I of the alternative σ factor, σ54. After phosphate release, the surface-exposed loops compact down towards the surface of the AAA+ domain, enabling relocation of the σ factor and formation of the open promoter complex [1214].

Structural features of NorR

Figure 1
Structural features of NorR

(A) Domain architecture of the bEBP NorR showing the N-terminal regulatory GAF domain (purple) containing a non-haem iron centre, the central ATPase-active domain (red) and the C-terminal DNA-binding domain (green) that contains an HTH motif. (B) Proposed model of the NO-sensing non-haem iron centre in the NorR regulatory domain. Structural model of the GAF domain of NorR based on the GAF-B domain of 3',5'-cyclic nucleotide phosphodiesterase [26] showing the iron centre (magenta) and proposed ligands Cys113, Asp96, Asp99, Arg75 and Asp131 (labelled). The Arg75 residue is the most likely to undergo ligand displacement upon NO binding. Also shown is the Arg81 residue, at the opposite end of an α-helix that also contains the Arg75 ligand. The model predicts that Arg81 is surface-exposed and well placed to make contact with the NorR AAA+ domain. (C) The σ54-interaction surface of the AAA+ domain of NorR is the target of GAF-mediated repression. Structural model of the AAA+ domain of NorR based on the NtrC1 structure [23] (PDB code 1NY5, chain A). The helices and loops (H3 and H4, L1 and L2) involved in nucleotide-dependent conformational changes in bEBPs are labelled in red. Residues that were substituted as a consequence of the PCR mutagenesis of the AAA+ domain are indicated. The Phe264 and Gly266 residues form part of the GAFTGA motif that contacts σ54.

Figure 1
Structural features of NorR

(A) Domain architecture of the bEBP NorR showing the N-terminal regulatory GAF domain (purple) containing a non-haem iron centre, the central ATPase-active domain (red) and the C-terminal DNA-binding domain (green) that contains an HTH motif. (B) Proposed model of the NO-sensing non-haem iron centre in the NorR regulatory domain. Structural model of the GAF domain of NorR based on the GAF-B domain of 3',5'-cyclic nucleotide phosphodiesterase [26] showing the iron centre (magenta) and proposed ligands Cys113, Asp96, Asp99, Arg75 and Asp131 (labelled). The Arg75 residue is the most likely to undergo ligand displacement upon NO binding. Also shown is the Arg81 residue, at the opposite end of an α-helix that also contains the Arg75 ligand. The model predicts that Arg81 is surface-exposed and well placed to make contact with the NorR AAA+ domain. (C) The σ54-interaction surface of the AAA+ domain of NorR is the target of GAF-mediated repression. Structural model of the AAA+ domain of NorR based on the NtrC1 structure [23] (PDB code 1NY5, chain A). The helices and loops (H3 and H4, L1 and L2) involved in nucleotide-dependent conformational changes in bEBPs are labelled in red. Residues that were substituted as a consequence of the PCR mutagenesis of the AAA+ domain are indicated. The Phe264 and Gly266 residues form part of the GAFTGA motif that contacts σ54.

Many bEBPs have an additional N-terminal regulatory domain (Figure 1A) that stringently controls the activity of the AAA+ domain either positively or negatively in response to various environmental cues [15]. NorR contains an N-terminal regulatory GAF (cGMP-specific and -stimulated phosphodiesterases, Anabaena adenylate cylases and Escherichia coli FhlA) domain that has been predicted to bind NO. It has previously been shown that an N-terminally truncated form of NorR, lacking the regulatory GAF domain (NorRΔGAF), is competent to activate transcription in the absence of NO [4,16]. Therefore NorR falls into a category of bEBPs in which the activity of the central AAA+ domain is negatively regulated by the N-terminal domain [17]. In the absence of NO, the GAF domain represses the activity of the AAA+ domain via interdomain repression [4]. The detection of the NO signal causes this repression to be relieved, allowing ATP hydrolysis by NorR and thus driving transcriptional activation. Other activators of σ54-dependent transcription that are controlled by similar mechanisms of interdomain repression include XylR [18,19], DmpR [20,21], DctD [22] and NtrC1 [23].

In addition to the central (AAA+) and regulatory domains, bEBPs also have a C-terminal HTH (helix–turn–helix) domain (Figure 1A) that binds to conserved enhancer-like sequences 80–150 bp upstream of the bacterial promoter. This ensures that activation of transcription occurs only at the specific promoter(s) with which the bEBP can associate. Interactions between the bEBP upstream of the promoter and the holoenzyme at the transcriptional start site are facilitated by DNA bending, assisted by IHF (integration host factor) [18,24].

In the present paper, we review recent developments in our understanding of the mechanism of NO-dependent activation of transcription by NorR. We address various aspects of this mechanism including: (i) a novel mechanism of NO sensing by the GAF domain, (ii) how the DNA-binding properties of the activator control NorR activity, and (iii) how the GAF domain negatively regulates the AAA+ domain.

Mechanism of NO sensing by NorR

In order to investigate the mechanism of NO sensing in NorR, EPR spectroscopy was carried out on whole cells of E. coli exposed to NO [25]. A new EPR signal was observed in the g=4 region only when the cells expressed NorR and were exposed to NO. This indicates that NorR contains a non-haem iron centre, since similar spectra have been observed for several non-haem iron enzymes when exposed to NO ([25] and references [1519] therein). This characteristic EPR signal was observed in cells expressing the isolated GAFNorR (GAF domain of NorR) but not in cells that expressed a form of the protein lacking the regulatory domain (NorRΔGAF), indicating that the non-haem iron centre is present within the N-terminal GAF domain. Purification and reconstitution of NorR and GAFNorR with ferrous iron gave identical NO spectra to those observed with whole-cell EPR, confirming that the NorR GAF domain contains the non-haem iron centre. This is the first example of a GAF domain using a transition metal as a mechanism of sensing and, to our knowledge, reveals a novel biological role for the activation of a non-haem iron centre to form a high-spin {Fe(NO)}7 (S=3/2) complex.

The spectroscopic features of this paramagnetic mononitrosyl iron complex suggest that the iron centre has distorted octahedral symmetry and is co-ordinated by five or six ligands within the GAF domain. In order to study the co-ordination of the iron centre in NorR, targeted mutagenesis was carried out at conserved residues within the regulatory domain [26]. As a result, five candidate ligands were proposed: Asp99, Asp131, Cys113, Arg75 and Asp96 (Figure 1B). Variant forms of NorR containing substitutions at these positions gave proteins that were unable to bind iron or did not exhibit the characteristic g=4 EPR signal after reconstitution in vitro. Therefore these residues are likely to have a role in iron co-ordination. These EPR data are in agreement with our hexa-co-ordinated structural model (Figure 1B). In the model, Asp96 is proposed to be a bidentate ligand. Although it is possible that a water molecule instead provides a sixth ligand, we believe this is unlikely since the iron-binding site appears to be solvent inaccessible. Although, arginine is not an ideal ligand for transition metals, examples have been reported elsewhere such as for biotin synthase [27]. The predicted hexa-co-ordination of the iron centre suggests that one of the five predicted ligands would need to be displaced in order to form the mononitrosyl iron complex. Arg75 is the most likely candidate to relinquish a binding site for NO and may also stabilize the NO-bound form of the iron through hydrogen bonding [26].

Role of enhancer-DNA in NorR-dependent activation of transcription

In previous work, purified NorR has been shown to bind to three sites in the norVW promoter region [28]. To assess the importance of each of these sites, the enhancers were individually altered from the consensus GT-(N7)-AC to GG-(N7)-CC and introduced upstream of norVlacZ promoter fusions on the E. coli chromosome. Disruption of any one of the three sites completely abolished the ability of NorR to activate transcription of norVW in vivo [29]. Biochemical experiments have demonstrated that the ATPase activity of NorR is dependent not only on the presence of NO but also on the enhancer DNA that contains the three NorR-binding sites [25]. In the absence of the regulatory GAF domain (NorRΔGAF), the requirement for the NO signal is relieved, but enhancer DNA is still required to stimulate activity. When any of the three binding sites was individually altered from the consensus, the enhancer-dependent ATPase activity of NorRΔGAF was significantly diminished [29]. Efficient open complex formation by NorR in vitro also required the three enhancer sites, consistent with the requirement for bEBPs to use the energy from ATP-hydrolysis to remodel the σ54-RNA polymerase holoenzyme [29]. The prerequisite for the three enhancer sites for transcriptional activation by NorR both in vivo and in vitro might reflect a requirement for three NorR dimers to assemble to form an inactive hexamer on the promoter DNA, given the dyad symmetry present at each of the sites. However, binding of NorR to a 21 bp sequence encoding one of the enhancer sites (NorR site 1) stimulated both the ATPase activity and oligomerization state to a certain extent, indicating that DNA binding per se promotes self-association and ATPase activity. Binding to a 66 bp DNA fragment that contained all three enhancer sites stimulated ATPase activity and oligomerization further, although not to the levels observed when a longer 266 bp fragment containing the intergenic region was used. This implies that the DNA flanking the enhancer sites has an important role in stabilizing the NorR oligomer, possibly by wrapping around the hexamer. In agreement with this, EMSAs (electrophoretic mobility-shift assays) revealed a significant increase in the affinity and co-operativity of binding when the longer DNA fragment was present. Furthermore, negative-stain electron microscopy revealed the formation of protein–DNA complexes with the expected diameter of a NorR hexamer in the presence of the 266 bp DNA, but not with the 66 bp or 21 bp fragments. Overall, these data support a model in which three NorR dimers bind to the enhancer sites, inducing conformational changes that stimulate the formation of a higher-order oligomer, most probably a hexamer. The results suggest that this higher-order NorR species is stabilized by extensive DNA interactions, possibly by wrapping around the hexamer to form a stable nucleoprotein complex.

Negative regulation of NorR activity

To investigate the mechanism of interdomain repression in NorR, we used a random mutagenic approach to search for mutations in the AAA+ domain that enable escape from GAF domain-mediated repression [30]. This approach generated mutant versions of NorR that were able to activate the transcription of a norV–lacZ fusion in vivo in the absence of NO, generated endogenously by the addition of potassium nitrite. In some cases, the NorR variants exhibited activity in the absence of NO signal that was similar to a truncated version of NorR lacking the GAF domain (NorRΔGAF). This phenotype indicates a complete bypass of the repression of AAA+ activity by the GAF domain. In structural models of the NorR AAA+ domain based on the crystal structure of the NtrC1 AAA+ domain (Figure 1C), the substitutions identified are located in H3 (helix 3), H4 (helix 4) or L1 (loop 1). This is the region of the AAA+ domain that undergoes nucleotide-dependent conformational changes before engagement with σ54 [9,12]. Significantly, two bypass mutations were identified in the GAFTGA motif, within surface-exposed loop 1. The G266D and G266N mutations within the second glycine of this motif enabled complete escape from the negative control exerted by the GAF domain. Furthermore, when additional substitutions were made to disrupt NO signalling at the iron centre, both GAFTGA variants retained the ability to activate transcription in vivo, suggesting that NO sensing is not required for their activity. The ability of these variants to activate transcription is surprising given the high conservation of the GAFTGA motif and that substitutions at this position are likely to affect the conformational flexibility of the loop.

In several bEBPs (e.g. NtrC1 and DctD), the N-terminal regulatory domain regulates the activity of the AAA+ domain by controlling the oligomerization state of the activator [22,23]. In order to further study how the Gly266 variants escape repression by the GAF domain, the variant proteins were purified using an N-terminal His-tag. DNA-binding studies demonstrated that both wild-type NorR and the G266D and G266N substitutions had similar affinities for the norVW promoter DNA (Kd ~100 nM) indicating that these Gly266 substitutions do not influence binding of NorR to the three enhancer sites. Furthermore, gel filtration studies indicated that, like NorR, the Gly266 variants eluted at a volume corresponding to a monomer or dimer in the absence of DNA, but formed stable higher-order oligomers in the presence of the promoter DNA. Analysis of eluted fractions by negative-stain electron microscopy showed the formation of particles of the size expected for a NorR hexamer. Furthermore, the ATPase activity of the substitutions was enhancer DNA dependent as is the case with wild-type NorR. Since the GAFTGA substitutions do not exhibit any changes in enhancer-dependent oligomerization, it seems unlikely that interdomain repression by the regulatory GAF domain is exerted by changing the oligomerization state of the AAA+ domain. Rather, the properties of the bypass mutations in the GAFTGA loop suggest that the regulatory domain negatively regulates the activity of the AAA+ domain by preventing access of the surface-exposed L1 and L2 loops to σ54 (Figure 2). This model is further supported by genetic suppression studies. Previous work identified the R81L substitution that results in partial escape from GAF-mediated repression [26]. Targeted mutagenesis at the Arg81 position confirms that this residue is critical in maintaining the mechanism of interdomain repression. Our structural model (Figure 1B) places it on the surface of the GAF domain, at the opposite end of an α-helix that also contains the Arg75 residue. Displacement of the Arg75 ligand upon NO binding would lead to significant conformational changes along this helix and so the Arg81 residue may have a key role in the transmission of the NO signal to the AAA+ domain. Significantly, hydrophobic changes at Arg81 are able to specifically suppress the escape phenotype of AAA+ bypass variants including the GAFTGA mutant G266D. This suggests that the GAF domain targets the GAFTGA motif to prevent interaction with σ54 in the absence of NO signal. Binding of NO to form the mononitrosyl iron complex is expected to lead to conformational changes in the GAF domain which release repression, allowing the GAFTGA motif to contact the σ factor.

Model of NorR-dependent activation of norVW

Figure 2
Model of NorR-dependent activation of norVW

(A) Binding of NorR to the norR–norVW intergenic region that contains the three NorR binding sites (1, 2 and 3, highlighted in red) is thought to facilitate the formation of a higher-order oligomer that is most likely to be a hexamer [29]. (B) Although the hexamer is bound to DNA (not shown), in the absence of NO, the N-terminal GAF domains (blue rectangles) negatively regulate the activity of the AAA+ domains (green circles) by preventing access of the surface-exposed loops to σ54. (C) In the ‘on’ state, NO binds to the iron centre in the GAF domain forming a mononitrosyl iron species. The repression of the AAA+ domain is relieved, enabling ATP hydrolysis by NorR coupled to conformational changes in the AAA+ domain. (D) During the nucleotide hydrolysis cycle, the surface-exposed loops (red) that include the GAFTGA motifs move into an extended conformation to allow σ54-interaction and remodelling.

Figure 2
Model of NorR-dependent activation of norVW

(A) Binding of NorR to the norR–norVW intergenic region that contains the three NorR binding sites (1, 2 and 3, highlighted in red) is thought to facilitate the formation of a higher-order oligomer that is most likely to be a hexamer [29]. (B) Although the hexamer is bound to DNA (not shown), in the absence of NO, the N-terminal GAF domains (blue rectangles) negatively regulate the activity of the AAA+ domains (green circles) by preventing access of the surface-exposed loops to σ54. (C) In the ‘on’ state, NO binds to the iron centre in the GAF domain forming a mononitrosyl iron species. The repression of the AAA+ domain is relieved, enabling ATP hydrolysis by NorR coupled to conformational changes in the AAA+ domain. (D) During the nucleotide hydrolysis cycle, the surface-exposed loops (red) that include the GAFTGA motifs move into an extended conformation to allow σ54-interaction and remodelling.

Conclusions

Our current model for the NO-dependent activation of norVW transcription by NorR is shown in Figure 2. The work reviewed in the present paper suggests that enhancer DNA induces conformational changes in NorR that allow the formation of an inactive hexamer. This is in stark contrast to bEBPs such as NtrC1 where the signal drives the process of oligomerization [23]. We suggest that in the case of NorR, the pre-formation of a NorR hexamer on enhancer DNA, ‘poised’ to respond to NO, allows the cell to rapidly respond to NO-induced stress. We have shown that this dedicated NO sensor detects the signal through the formation in a novel mononitrosyl complex at the non-haem iron centre of the regulatory domain. Furthermore, we have identified a novel mechanism of interdomain repression in which the regulatory domain targets the σ54-interaction surface in the absence of NO to prevent ATP hydrolysis.

Enzymology and Ecology of the Nitrogen Cycle: A Biochemical Society Focused Meeting held at University of Birmingham, U.K., 15–17 September 2010. Organized and Edited by Jeff Cole (University of Birmingham, U.K.), Rosa María Martínez-Espinosa (University of Alicante, Spain), David Richardson (University of East Anglia, Norwich, U.K.) and Nick Watmough (University of East Anglia, Norwich, U.K.).

Abbreviations

     
  • AAA+

    ATPase associated with various cellular activities

  •  
  • bEBP

    bacterial enhancer-binding protein

  •  
  • GAF

    cGMP-specific and -stimulated phosphodiesterases, Anabaena adenylate cylases and Escherichia coli FhlA

  •  
  • GAFNorR

    GAF domain of NorR

  •  
  • HTH

    helix–turn–helix

  •  
  • NO

    nitric oxide

Funding

This work was supported by a research grant from the Biotechnology and Biological Sciences Research Council to R.D. [grant number BB/D009588/1].

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1278
-
1288
)