Common to all of the nitrate nitrite porter family are two conserved motifs in transmembrane helices 5 and 11 termed NS (nitrate signature) 1 and NS2. Although perfectly conserved substrate-interacting arginine residues have been described in transmembrane helices 2 and 8, the role of NSs has not been investigated. In the present study, a combination of structural modelling of NrtA (nitrate transporter from Aspergillus nidulans) with alanine scanning mutagenesis of residues within and around the NSs has been used to shed light on the probable role of conserved residues in the NSs. Models show that Asn168 in NS1 and Asn459 in NS2 are positioned approximately midway within the protein at the central pivot point in close proximity to the substrate-binding residues Arg368 and Arg87 respectively, which lie offset from the pivot point towards the cytoplasmic face. The Asn168/Arg368 and Asn459/Arg87 residue pairs are relatively widely separated on opposite sides of the probable substrate translocation pore. The results of the present study demonstrate the critical structural contribution of several glycine residues in each NS at sites of close helix packing. Given the relative locations of Asn168/Arg368 and Asn459/Arg87 pairs, the validity of the models and possible role of the NSs together with the substrate-binding arginine residues are discussed.
- alanine scanning
- helix packing
- major facilitator superfamily (MFS)
- nitrate transporter
- structural model
Nitrate is a relatively simple small inorganic molecule with an ionic radius of only 1.96 Å (1 Å=0.1 nm), but it has a dual character of considerable biological positivity or negativity. As Dr Jekyll, nitrate acts as a major nitrogen source in natural environments for numerous bacteria, fungi, algae and plants, including crop plants, and it is the nutrient that most frequently limits their growth (for reviews, see [1,2] and references therein). In addition, vast quantities of nitrogen-based fertilizers, rapidly converted into nitrate by soil oxidizing bacteria , are widely used in agriculture to sustain maximum crop yields, thereby satisfying the needs of our increasing human population and its food demands. Nitrate, in this beneficial mode, is assimilated to ammonium and catalytically transformed into amino acids for protein synthesis and growth of these organisms. Furthermore, vegetable dietary nitrate in the human oral cavity and gut may be utilized by nitrate-reducing symbiotic bacteria to provide protection against pathogenic bacterial species ([4,5] and references therein). In Mr Hyde mode, nitrate is a serious water pollutant, readily leaching from soils, contributing to eutrophication of natural water systems with resultant loss of biodiversity and simultaneously lowering nitrogen availability to environment land as well as crop plants . Secondly, a derivative of nitrate, nitrous oxide, emitted from both anthropogenic and ecological activities, is a major greenhouse gas . Finally, there are human and animal health concerns as carcinogenic N-nitrosamines also derived from nitrate may be generated by bacteria in the human gut ( and references therein).
The transport of nitrate across membrane barriers by several classes of proteins, including the ABC transporters , the low-affinity transporters  and the high-affinity transporters , has been studied by a number of research groups. The high-affinity Nrts (nitrate transporters) are particularly important, being present in all kingdoms except animal. Following the description of the first gene encoding a high-affinity NrtA (Nrt from Aspergillus nidulans) , it became clear that such transporters belong to the NNP (nitrate/nitrite porter) family [12,13] a subfamily forming a distinct cluster of the largest secondary transporter family, the MFS (major facilitator superfamily) (Transport Classification Database number TC 2.A.1). This superfamily comprises a wide range of functionally diverse transporters , most of which possess 12 TMs (transmembrane α-helices). Although the determination of the structure of a high-affinity Nrt has proven recalcitrant to crystallography, the structures of several prokaryotic MFS transporters, namely LacY (lactose transporter from Escherichia coli) , GlpT (phosphate/glycerol-3-phosphate antiporter from E. coli) , EmrD (multidrug transporter from E. coli) , FucP (fucose transporter from E. coli)  and PepTSo (oligopeptide transporter from Shewanella oneidensis) , have been solved. However, although the location of residues and motifs can be more accurately mapped from comparison with these 3D (three-dimensional) crystal structures than from 2D (two-dimensional) secondary structure models, the transport mechanisms involved may still be obscure without detailed biochemical information.
Previous studies of NrtA (and the Escherichia coli nitrate transporter NarK) have indicated that two perfectly conserved arginine residues, Arg87 and Arg368 in TM2 and TM8 respectively, are essential and most probably represent the substrate-binding site for nitrate in the NNP family proteins [20,21]. In addition, second-site suppressor results indicated that at least one of these, Arg87, was located in close proximity to an asparagine residue, Asn459, in TM11. Asn459 resides in a region of the transporter that is recognized as a conserved motif present in all NNP family transporters, and is not observed in any other class of MFS proteins . Nor is this motif apparent in the MFS-related Nrts exemplified by CHL1 (dual-affinity nitrate transporter of Arabidopsis thaliana) (NRT1.1), a protein belonging to the NRT1/PTR family (Transport Classification Database number TC 2.A.17) . This is despite the ability of the phosphorylated form of CHL1 to carry out high-affinity nitrate transport. In NNP proteins, this conserved residue stretch has been referred to as the NS (nitrate signature) of which there are two copies within NrtA, located in TM5 and TM11 (Figure 1).
Towards understanding the importance of NS1 and NS2 and their role in the structure and function of this anion transporter, we have carried out systematic alanine scanning in-vitro mutagenesis on these motifs. Specifically, we have altered NS1 and NS2 amino acids, as well as several residues N- or C-terminal to NS1 and NS2, to alanine and biochemically characterized the resulting mutant strains. In addition, we have developed models for the nitrate transporter on the basis of the crystal structures of GlpT (inward-facing) and FucP (outward-facing) [16,18] to complement the analysis of the mutant information, as well as allowing a re-evaluation of previous mutant data  and assessment of possible mechanisms for the transport process by NNP proteins. Our analysis indicates that the NSs fulfil several functions in nitrate transporters: conserved glycine residues are required for positioning of helices as well as for close helix packing and flexibility, whereas Asn168 in NS1 and Asn459 in NS2 may have a more direct role in nitrate transport.
Microbial strains and media
Standard procedures were used for propagation of plasmids, as well as for subcloning and maintenance of plasmids within E. coli strain DH5α. A. nidulans strains used in this study were: (i) the standard wild-type (with regard to nitrogen metabolism) biotin auxotroph, strain biA1, (ii) the double-deletion mutant nrtA747 nrtB110 (disrupted in both nrtA and nrtB), strain T110 and (iii) the triple-mutant strain harbouring nrtA747 nrtB110 mutations as well as the arginine auxotrophic marker argB2, strain JK1060. Standard Aspergillus growth media and handling techniques were as described previously . Shake flask cultures for nitrate-uptake assays were grown in liquid minimal medium (, and as modified in ).
Genetic transformation procedure
The A. nidulans transformation procedure to obtain single-copy integration at the argB locus was essentially as described previously (reviewed in  and references therein). For each mutation, approximately ten arginine auxotrophic transformant strains were analysed by Southern blotting to identify a single nrtA gene copy integration at argB.
Generation of amino acid replacement constructs by PCR overlap extension was as described previously  with all constructs being verified by DNA sequencing. The constructs all included a sequence resulting in C-terminal fusion of a V5 tag  to the NrtA protein. BamHI digestions of genomic DNA were analysed by Southern blotting following transformation of the constructs as described previously . For PCR amplification to verify the sequence of single-copy alanine replacement mutants, DNA was isolated from 50 mg of pressed wet mass of mycelium suspended in 500 μl of breaking buffer [2% (v/v) Triton X-100, 1% (w/v) SDS, 100 mM NaCl, 10 mM Tris/HCl, pH 8.0, and 1 mM EDTA, pH 8.0], to which was added 500 μl of chloroform/3-methylbutan-1-ol (24:1) and 300 μl of 0.5 mm-diameter glass beads (BioSpec). Mycelium was homogenized using a FastPrep 24 homogenizer at speed 4.5 for 20 s followed by centrifugation at 16000 g for 10 min at room temperature (25°C). The upper phase was transferred to a new 1.5 ml microcentrifuge tube, and 2 vol. of ice-cold 96% (v/v) ethanol were added and mixed. DNA was pelleted by centrifugation at 16000 g for 10 min, washed with 70% (v/v) ethanol, dried and resuspended in 50 μl TE buffer (10 mM Tris and 0.1 mM EDTA, pH 8.0) containing 10 μg/ml RNase A. A 0.5 μl portion of the DNA was used in 50 μl PCR volumes using Phusion High Fidelity DNA polymerase (New England Biolabs) and the primers MUTF and MUTR2 .
Sequence logos were generated from an alignment of 500 prokaryotic and 173 eukaryotic proteins identified in a BlastP search using the E. coli nitrate transporter NarU sequence as the query. Multiple alignments were generated using MAFFT  at the European Bioinformatics Institute (http://www.ebi.ac.uk) and entered into Jalview , from which the required sequences were extracted and entered into Weblogo (http://weblogo.berkeley.edu). Colour information was discarded from the Weblogo output and logos were presented in greyscale.
These were performed on cultures grown in minimal medium with 5 mM urea as the nitrogen source for a total of 6.5 h at 37°C with induction of the transporter by addition of 10 mM sodium nitrate 100 min prior to the assay, as described previously . Assays measuring depletion of nitrate from the medium after incubation for 20 min at 37°C were carried out on at least three independently grown cultures for each strain and expressed as nmol of nitrate removed from the medium/min/mg of dried mycelium.
Protein expression analysis
For immunological detection, cultures were grown as for the net-nitrate-uptake assays and crude membrane preparations were made from 50 mg of pressed wet mass of mycelium. Mycelium was suspended in 500 μl of ice-cold extraction buffer consisting of 10 mM sodium orthophosphate, 200 mM sodium chloride and 10% (v/v) glycerol, pH 7.0, to which was routinely added fresh 0.1 mM PMSF and 1 mM benzamidine. As mentioned in the Discussion section, some preparations were tested with additional complete protease inhibitor cocktail (Roche) in the extraction buffer. A 300 μl portion of 0.5 μm glass beads was added and cells were disrupted by homogenization at speed 4.5 for 20 s in a FastPrep 24 homogenizer. After 2 min on ice, samples were centrifuged at 16000 g for 1 min at room temperature. The supernatant fraction was centrifuged at 4°C for 45 min at 25000 g and the resulting crude membrane pellet was resuspended in 50 μl of ice-cold extraction buffer. Samples of 1 μl, equivalent to approximately 50 μg of protein, were run on a 4–12% NuPAGE gel (Invitrogen), blotted on to Invitrolon membrane (Invitrogen) as described in the manufacturer's instructions and NrtA protein was detected using an anti-V5–HRP (horseradish peroxidase) antibody (Invitrogen) and an ECL Plus Western Blot Detection System (GE Healthcare). At least three independently grown cultures were analysed for each strain and representative results are shown.
Generation of 3D NrtA models
Owing to the low sequence similarity between MFS proteins, molecular structural models for NrtA were built using the threading, or fold recognition, servers Phyre  and iTasser . Models were returned on the basis of MFS templates, including GlpT, LacY, FucP and PepTSo. The similarity in the models based on different templates and from the two different servers gave confidence that the results were credible models. The models based on GlpT (PDB code 1PW4) and FucP (PDB code 3O7Q) were selected as representatives of the open-inward and open-outward states respectively. The large intracellular loop between TMs 6 and 7 from Thr240 to Phe291 was removed from these models. PyMOL (http://www.pymol.org) software was used to analyse the models and produce structural cartoons (Figure 3).
Amino acid sequence comparative analysis of NS1 and NS2
Figure 2 provides a graphical display of sequence conservation generated from multiple amino acid sequence alignments obtained from putative nitrate transporters of prokaryotes and eukaryotes where the height of a residue reflects its frequency at that position. Within these displays can be seen the sequences originally identified as the NS motifs of NrtA [12,13]. However, with the increasing availability of genome sequences and the consequent rise in predicted nitrate transporter proteins, such alignments permit a reassessment and redefinition of the most highly conserved residues in and around NS1 and NS2. Clearly from Figure 2, the prokaryotic signatures show a higher level of sequence divergence (i.e. greater ‘choice’ of residues) than eukaryotes at many of the positions within the alignment, possibly as a consequence of the far larger number of prokaryotic sequences available, with conservation in NS1 being stronger than NS2. Nevertheless, certain residues appear almost invariant, such as the glycine at position 1 of the alignment, glycine at position 10 of NS2 and the GNXG at positions 11–14 of NS1. These latter residues and the position 1 glycine are also highly conserved in the eukaryotic signatures as well as others such as asparagine at position 4 of NS1 and glycine at position 5 of NS2, glycine at positions 9 in NS1 and 8 in NS2, and glycine residues at positions 15 and 16 of NS1.
3D models of NrtA
The actual sequences of the NrtA signatures relative to the alignment positions are shown in Figure 2. Thus glycine 1 of the alignment is equivalent to Gly157 of NS1 and Gly448 of NS2. These and other highly conserved residues from the eukaryotic sequences in Figure 2 can be observed in the NS1 and NS2 sequences of NrtA. The 3D models of NrtA in Figure 3 are based on the crystal structures of the open-inward GlpT (Figures 3A, 3C and 3E) and open-outward FucP (Figures 3B, 3D and 3F), conforming to the alternating access mechanism. Residues Gly157 and Gly448 locate near the N-terminus of their respective TMs 5 and 11. Directly above these in the helices are Ser161 and Gly165 in TM5 and Gly452 and Gly456 in TM11. In the open-outward FucP-based model (Figures 3B and 3D), these residues are tightly packed against other TMs in the ‘closed’ portion of the molecule. Facing the lumen within the protein in which the nitrate-binding residues Arg87 and Arg368 are located (Figures 3A–3D), the first turn of TM5 contains Asn160 that is perfectly conserved in the eukaryotic NS1 sequences. Two helix turns above this lies the Asn168 residue of the NS core GNXG motif (Figures 3A and 3B). In TM11, the highly conserved Gly452 is in an approximately equivalent position to Asn160, two helix turns below the core motif asparagine residue, Asn459 (Figure 3C and D). Other conserved glycine residues are placed around the helix close to Asn168 in TM5 (Gly167, Gly170, Gly171 and Gly172) and Asn459 in TM11 (Gly455, Gly458, Gly461 and Gly462) (Figures 3A–3D), whereas relatively bulky side chains such as Leu166 in TM5 and Phe457 and Leu460 in TM11 are on the opposite face of the helix from Asn168 and Asn459 respectively. The models in both inward- and outward-facing conformations show the position of Asn168 and Asn459 to be approximately central in the protein, with Arg87 and Arg368 roughly two-thirds towards the cytoplasmic side (Figures 3A–3D). Significantly, within the central lumen, both models show close proximity of Asn168 to Arg368 and Asn459 to Arg87 (Figures 3E and 3F), the latter consistent with the functional replacement reported previously .
Alanine scanning of NS1
Gly157 to Thr174 of NS1 in TM5 were individually changed to alanine, including residues of the first NS, as well as several residues flanking the signature. Natural alanine residues at positions 159, 163, 164 and 169 were left unaltered. Alanine replacement mutant strains were examined for: (i) growth ability on nitrate as the sole nitrogen source, (ii) net nitrate transport where growth was observed on nitrate and (iii) protein expression levels by Western blot analyses.
(i) Growth phenotypes
Alanine scanning of all four highly conserved residues within NS1 in mutants G165A, G167A, N168A and G170A resulted in complete loss of the ability to grow on nitrate at 37°C, indicating loss of function (Table 1). Similarly, alanine replacement of residues conserved only in eukaryotes permitted slight growth (G171A), whereas G172A failed to grow. In contrast, mutants with alanine replacements of residues flanking conserved residues and mainly poorly conserved, residues T158A, N160A, S161A and L162A (N-terminal to the NS1 core) as well as I173A and T174A (C-terminal to the NS1 core), retained wild-type or near wild-type growth. Finally, L166A (located in the NS1 core) showed intermediate growth, and G157A (at the N-terminus of TM5) led to loss of function as judged by growth response. Growth responses were also tested on nitrate as the sole nitrogen source at 25°C to assess the possibility of mutant temperature sensitivity. For most mutants, growth at 25°C paralleled that at 37°C, with the exception of mutant G171A, which grew well relative to the wild-type at 25°C despite showing very poor growth at 37°C (results not shown).
(ii) Net-nitrate-uptake activity
Assays were carried out for mutants that showed growth on nitrate (Table 1). As expected from the growth phenotypes, net-nitrate-uptake activity of the mutants T158A, S161A, L162A, I173A and I174A, located before and after the core signature, was similar to the wild-type level. The exception to this was the strain possessing alanine substitution of residue, Asn160, a very highly conserved residue in the eukaryotic Nrts. This mutant showed a notable decrease in activity compared with the wild-type. Change of the core signature residue Gly171 to alanine resulted in substantially reduced net-nitrate-uptake activity correlating with the poor growth of this mutant. Mutant L166A, concomitant with slightly better growth on nitrate than G171A, displayed an intermediate rate of nitrate uptake. Mutant G165A was chosen for assay as a representative of those mutants unable to grow on nitrate to check that the level of activity was indeed as low as the negative control strain T110.
(iii) Protein expression levels
Western blot analyses showed that the wild-type strain T454 (Figure 4) has a single band of 45 kDa, whereas no protein was detected in the negative control strain T110 (Figure 4B). Virtually no NrtA protein was observed in crude membranes prepared from mutants in the highly conserved residues G157A, N168A and G172A (Figure 4A). Strains G165A, G167A and G170A showed greatly reduced levels of NrtA and possessed a second band of approximately 41 kDa. Although the total amount of NrtA protein detected in the crude membranes from mutants L166A and G171A was higher than for G165A, G167A and G170A, both bands of 45 kDa and 41 kDa were observed. The smaller band of 41 kDa is suggestive of proteolysis, but this is unlikely to be due to digestion during protein isolation, since experiments in which a protease inhibitor cocktail was added to the extraction buffer made no difference to the appearance of the preparation in Western blots (results not shown). Nor is the smaller band likely to be the result of inherent protein instability causing aberrant migration in the gel, as trial experiments on the NrtA protein solubilized from crude membranes of F457A to the action of trypsin showed no increased susceptibility over the wild-type protein (results not shown). Therefore proteolysis of the mutant proteins most probably occurs during trafficking to the membrane or following insertion into the membrane. In this regard, it should be noted that the preparations examined were crude membrane fractions. Hence, in those mutants with reduced or no activity, NrtA protein observed in Western blots may be located in an endomembrane compartment other than the plasma membrane. Mutants N-terminal (T158A, N160A, S161A and L162A) and C-terminal (I173A and T174A) to the core possessed a single wild-type band of approximately 45 kDa.
Alanine scanning of NS2
Residues Gly448 to Phe465 in TM11 covering the NS2 core region as well as flanking regions were altered to alanine and mutant strains were examined as for NS1 mutants.
(i) Growth phenotypes
In contrast with many of the mutants in the NS1 region, most alanine substitution mutants in the NS2 area retained the ability to grow on nitrate at 37°C as a sole nitrogen source (Table 1). Only N459A completely lacked growth. Reduced growth on nitrate relative to the wild-type was observed for mutants of highly conserved residues, G448A and G452A, and of those within the NS2 core region, G455A, G456A, F457A, G458A, L460A, G461A and G462A. Other alanine substitution mutants, I449A, V450A, S451A, M453A and V454A, N-terminal to the core residues, and I463A, I464A and F465A, C-terminal to the core residues, showed growth comparable with the wild-type. At 25°C, most mutants displayed a similar pattern of growth on nitrate as at 37°C, except for the mutants G448A and G455A, which appeared to grow as well as the wild-type strain at 25°C (results not shown).
(ii) Net-nitrate-uptake activity
Concomitant with the growth phenotypes, net nitrate uptake was observed for most of the mutants in the NS2 region, with those displaying wild-type growth on nitrate having uptake rates of approximately 8 nmol nitrate/min/mg, similar to the wild-type (Table 1). Intermediate values of between 1.2 and 4.7 nmol nitrate/min/mg were obtained for those mutants that grew less well on nitrate.
(iii) Protein expression levels
Mutants flanking the core region, I449A, V450A, S451A, M453A and V454A (N-terminal) and I463A, I464A and F465A (C-terminal), showed a single band of 45 kDa similar to the wild-type (Figure 4B). Mutants in the core region showed, in some cases, in total almost wild-type protein levels (G455A, G456A, F457A, G458A, G461A and G462A) or reduced expression (L460A) and all possessed a second band of approximately 41 kDa. Similarly, crude membranes of mutants of highly conserved residues Gly448 and Gly452 altered to alanine showed good expression (G448A) or poor expression (G452A) and two bands of 45 and 41 kDa. Almost no NrtA expression was observed in the N459A strain.
The clear deleterious effects of alanine scanning mutagenesis on residues within the NSs underline the importance of these conserved motifs as structural features necessary to facilitate the structure and/or function of the NrtA protein. It is interesting that most of the residues showing severely reduced protein expression occur in NS1 suggesting that, although they are apparent symmetry partners in the structural models, TM5 is less able to accommodate change than TM11. A notable feature of the NSs is the large number of glycine residues. Both NSs contain serial sequences of glycine residues, two or three together, spacing that suggests flexibility and/or rotation of the strand may be required for optimal function. Indeed, molecular dynamics simulations have highlighted the highly flexible nature of TM5 and TM11 in both LacY and GlpT [33,34]. The larger side chain of alanine compared with glycine substantially reduces the possible range of bending and/or rotation of a polypeptide strand . Thus the observation that substitution of glycine by alanine in the equivalent TMs of NrtA leads to proteins of low or no activity, along with poor folding of the whole protein (as evidenced by the 41 kDa band observed in Western blots of several mutants), is consistent with the need for structural flexibility exhibited in the LacY and GlpT simulations.
In addition to flexibility, a key property of glycine residues located within TMs is that their small side chain allows close helix–helix interaction, exemplified by the GXXXG motif of glycophorin A and other proteins [36,37]. In NrtA, two contiguous and overlapping GXXXG/SXXXG motifs are present at the cytoplasmic ends of both TM5 and TM11 at residues 157–165 in TM5 and residues 448–456 in TM11. These are reminiscent of glycine zipper motifs  and, as expected for such motifs, are well conserved in nitrate transporters (Figure 2), with perhaps the exception of TM11 of the prokaryotic proteins. In nitrate transporters, therefore, it is likely that the contiguous cytoplasmic GXXXG/SXXXG motifs are required for tight helix packing to close the cytoplasmic end of the protein in the outward-facing conformation, as suggested for similar motifs in OxlT (oxalate/formate antiporter from Oxalobacter formigenes) and GlpT [39,40]. In the open-outward model (Figures 3B and 3D), the glycine residues of the GXXXG/SXXXG motifs all reside on the same face of the helix as the asparagine residues Asn168 and Asn459 in their respective TMs in an orientation suggesting directional close packing of TM5 towards TM8 (the location of substrate-binding residue Arg368) and TM11 towards TM2 (the location of substrate-binding residue Arg87). The preponderance of glycine should allow close and dynamic association of the nitrate-binding residues Arg87 and Arg368 with Asn459 and Asn168 respectively. Indeed, in the helix turn immediately C-terminal (toward the cytoplasmic face) to Arg87 in TM2 and Arg368 in TM8 are one or two, respectively, very highly conserved glycine residues (Gly91 in TM2, and Gly371 and Gly372 in TM8) whose location could allow very close proximity of Asn459 and Asn168 respectively.
In NrtA, GXXG motifs encompass the conserved asparagine residues Asn168 and Asn459, roughly in the centre of TM5 (residues 167–170) and TM11 (residues 458–461) (Figures 3A–3D). In both the inward-facing and outward-facing models of NrtA, the glycine residues in these motifs are positioned such that those in TM5 abut TM1 and those in TM11 are closest to TM7, just at the points of crossover of TM5 with TM1 and TM11 with TM7. These GXXG motifs are well conserved and often immediately followed by up to three residues of small side chain volume, principally glycine residues in eukaryotic nitrate transporters (Figure 2). We propose that these central GXXG motifs at positions 167–170 in TM5 and 458–461 in TM11, and their associated glycine residues Gly171 and Gly172 (TM5) and Gly462 (TM11), are critical to inter-helix crossover, thus permitting juxtaposition of helices carrying the residues for substrate transport.
Prominent in both NS1 and NS2 are the conserved asparagine residues that have previously been shown to be irreplaceable (Asn168) or to allow only very conservative change to glutamine (Asn459) . Leu166 or Phe457 are located on the opposite side of the helices from Asn168 and Asn459 respectively, and so a change to alanine might not be expected to alter substantially transport function and permit the observed level of activity. The proximity of Asn459 to the perfectly conserved Arg87 in TM2, previously inferred by complementary substitutions , can be seen in the models of NrtA, as can the equivalent positioning of Asn168 close to Arg368 (Figure 3). Given the evidence indicating that Arg87 and Arg368 are substrate-binding residues , the proximity of the asparagine residues in the NSs to the respective arginine side chains, their position right at the apparent location of the hinge between the two alternating access conformations and the propensity of asparagine residues for hydrogen bond formation, all suggest a potentially important function in nitrate transport for Asn168 and Asn459. However, the complete loss of detectable protein by Western blot analysis for the two mutants N168A and N459A, demonstrating an important structural role for these asparagine residues, precludes interpretation of a specific functional role in nitrate transport.
One common theme in MFS anion transporters is the requirement for two positively charged residues in the co-ordination of negatively charged substrates. In the best-studied examples, namely GlpT and OxlT, these positively charged residues are located at or close to the proposed pivot point (according to the model of alternating access) of the transporters roughly at the centre of the protein. Thus the GlpT antiporter possesses two arginine residues, Arg46 and Arg269 located in TM1 and TM7 respectively, which have been proposed to interact directly with phosphate or the phosphate moiety of glycerol-3-phosphate . Molecular dynamics simulations indicate that this binding by the arginine residues is not simultaneous, but occurs initially to Arg46 and probably sequentially to Arg269 during the substrate translocation process . Combined biochemical evidence and structural modelling (based on the GlpT structure) predict that in the OxlT protein, Arg272 and Lys355 in TMs 8 and 11 respectively form the binding site for the substrate for oxalate [41,42]. Therefore in each of these proteins the anion-binding residues are positioned within 10 Å of each other such that the side chains can interact with substrate, either simultaneously or in sequence, effectively forming the substrate-selective binding site. NrtA likewise has two positively charged residues, Arg87 and Arg368, implicated in nitrate binding. However, in contrast with GlpT and OxlT, relative spacing of residues based on the inward-open (GlpT) and outward-open (FucP) models clearly shows the comparatively wide separation of approximately 15 Å between Arg87 and Arg368. This distance is far greater than would support the simultaneous co-ordination of the small anion, nitrate, with an ionic radius of just 1.96 Å . Similarly, the residue side chains are modelled such that sequential binding would also appear improbable with this separation. In addition, from the models of NrtA, the positions of Arg87 and Arg368 in the protein are not central, but offset towards the cytoplasmic face. Undoubtedly, the distances obtained from models based on templates with low amino acid sequence similarity, such as that of NrtA for GlpT or FucP, will be subject to inaccuracies in assigning precise positions for individual residues. Nevertheless, experimental data obtained with numerous MFS transporters of diverse substrates such as the Saccharomyces cerevisiae phosphate transporter Pho84 , the E. coli multidrug transporter MdfA , the human organic anion transporter 1 hOAT1 , the S. cerevisiae monocarboxylate transporter Jen1p  and the sialic acid transporter Sialin  have shown broad agreement with the respective models based on GlpT and supported the overall structural conservation within the MFS. In order to maintain this overall structure, but retain a single substrate-binding site, the close packing afforded by the presence of glycine residues conserved in nitrate transporters may allow the substrate-binding arginine side chains to be substantially closer (approximately 7 Å) than the basic modelling suggests. NrtA and other nitrate transporters would therefore be markedly more compact proteins than hitherto studied MFS transporters. Alternatively, there may be an active intermediate structure where conformational change brings Arg87 and Arg368 transiently close enough together to bind a nitrate ion simultaneously or to pass nitrate from one to the other in sequence.
Shiela Unkles and James Kinghorn proposed the study, and conducted experiments and analysis; Eugenia Karabika, Vicki Symington and Shiela Unkles performed the molecular biology work, designed the mutants, made the plasmids and generated the mutants; Vicki Symington and Shiela Unkles assayed the mutants for net nitrate uptake; Eugenia Karabika, Vicki Symington and Naureen Akhtar evaluated protein expression; Jennifer Cecile, Duncan Rouch and Brett Cromer designed models. All of the authors participated in discussion of the results and wrote the paper.
This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) [grant number BB/E012213/1 (to S.E.U.)]. V.F.S. acknowledges the support of a BBSRC studentship, and N.A. the support of the Higher Education Commission (HEC) of Pakistan and University of the Punjab, the Charles Wallace Pakistan Trust, the British Federation of Women Graduates and the Leche Trust U.K.
Abbreviations: CHL1, dual-affinity nitrate transporter of Arabidopsis thaliana; 3D, three-dimensional; FucP, fucose transporter from Escherichia coli; GlpT, phosphate/glycerol-3-phosphate antiporter from Escherichia coli; LacY, lactose transporter from Escherichia coli; MFS, major facilitator superfamily; NNP, nitrate/nitrite porter; Nrt, nitrate transporter; NrtA, Nrt from Aspergillus nidulans; NS, nitrate signature; OxlT, oxalate/formate antiporter from Oxalobacter formigenes; PepTSo, oligopeptide transporter from Shewanella oneidensis; TM, transmembrane α-helix
- © The Authors Journal compilation © 2012 Biochemical Society