DNQX-mediated hypocotyl elongation is competitively inhibited by glycine and glutamate
Plant GLRs have been implicated in the control of an important component of light-mediated development – hypocotyl elongation (Brenner et al., 2000; Lam et al., 1998). As has been shown previously (Lam et al., 1998), plants grown in the presence of DNQX have elongated hypocotyls (Figure 4a). If DNQX elicits this response by antagonising plant GLRs, then simultaneous application of GLR agonists should reverse this effect. Both glutamate and glycine reversed DNQX-induced hypocotyl elongation in a concentration-dependent fashion (Figure 4). Glycine was at least as effective as glutamate at reversing the DNQX effect. Glycine and glutamate functioned synergistically when applied together with DNQX to seedlings, functioning at significantly lower concentrations than when they were used alone (Figure 4b).
Figure 4. Variation in hypocotyl elongation in response to application of glutamate (Glu), glycine (Gly), DNQX or combinations thereof.
(a) Arabidopsis seedlings were grown in medium containing the effectors at the concentrations indicated, and changes in hypocotyl elongation were visualised.
(b) The quantitative response to the effectors was determined. Values represent the mean of a minimum of 15 seedlings. Pairwise t-tests were used to compare the mean of each treatment against the mean for the seedlings grown in MS plus DNQX (★: significant difference; P < 0.0001) or MS alone (○: no significant difference; P < 0.0001). Error bars show the SE of the mean.
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While DNQX pre-treatment of seedlings completely inhibited ligand-gated changes in [Ca2+]cyt in aerial tissues, as monitored by aequorin (Figure 3b), it is clear that growth of seedlings in medium containing a mixture of DNQX and amino acids allows for competition to occur between the agonists and the antagonist, so that the effect is not absolute but concentration-dependent. It is important to note that the experiments with aequorin document the ability of DNQX pre-treatment to block the immediate change in [Ca2+]cyt, and do not indicate the long-term effect on [Ca2+]cyt, gating by the three ligands acting together. The growth of plants in a combination of DNQX with the amino acids would almost certainly result in a dynamic competition for the binding site by the inhibitor and the two agonists. Furthermore, long-term exposure of plants to DNQX may alter the abundance of GLRs, and thereby alter plant responsiveness to the agonists. In animals, the abundance of GLRs is affected by the extent to which a neuron is stimulated by neurotransmitters. The greater the stimulation, the lower the abundance of GLRs and vice versa (Platenik et al., 2000; Scannevin and Huganir, 2000; Sheng and Kim, 2002). Similarly, continual exposure of plants to DNQX and concomitant decreases in GLR stimulation may lead to an increase in the abundance of GLRs, which would, in turn, enhance plant responsiveness to any glutamate and glycine that is present. This may account for the ability of glutamate and glycine to reverse the effects of DNQX when plants are grown in a mixture of these ligands. Finally, it may also be that the combined action of glutamate and glycine reverses the inhibitory effects of DNQX through an alternative signalling mechanism, which may be calcium-independent and which could account for the apparent differences between the calcium influx data and the hypocotyl elongation data.
Molecular modelling shows binding of plant GLRs to glutamate, glycine and DNQX
As was the case in previous studies, the results described herein provide circumstantial evidence for the interaction between plant GLRs and their putative ligand, glutamate. Beyond this, however, the current results provide more comprehensive evidence in support of the hypothesis that plant GLRs function like their mammalian counterparts in that the gating of [Ca2+]cyt is modulated by both glutamate and glycine, and that this is inhibited by DNQX. Furthermore, the results presented herein show that this signalling mechanism may be involved in the control of hypocotyl elongation. Nevertheless, it remains to be determined if any of the compounds in question actually interact with plant GLRs. As an important first step in determining if glutamate, glycine and DNQX are able to bind to plant GLRs, a molecular modelling approach was employed.
The structure of the extracellular ligand-binding region of an A. thaliana GLR subunit (AtGLR) was modelled on the crystal structure of the analogous region of a Rattus norvegicus GLR (PDB ID 1FTJ; Armstrong and Gouaux, 2000). The structure of the rat GLR and its interaction with its ligands was empirically determined by crystallography as well as detailed functional analyses of GLR-ligand interactions (Armstrong and Gouaux, 2000; Armstrong et al., 1998; Yoneda and Ogita, 1991). Using a multiscale docking algorithm that provides accurate predictions of protein–ligand interactions (Glick et al., 2002a,b,c), the nature of the interaction of the plant GLRs with glutamate and glycine was explored. The A. thaliana GLR, AtGLR2.9, was chosen for the modelling study as it exhibited the greatest sequence similarity to the R. norvegicus GLR, particularly in those regions implicated in ligand binding (Figures 5 and 6).
Figure 5. Multiple sequence alignment of GLR sequences.
The peptide sequences (and their corresponding accession numbers) that were used in the alignment included: R. norvegicus GLRs, RatGlurB (CAA38465), RatKain1 (AAA02873), NR2a (AAC03565), NR2b (AAA41714), NR2c (AAA41713), NR2d (AAC37647), NR1a (AAA16366) and NR3b (AAL69893); Homo sapiens GLR, NR3a (AAL40734) and A. thaliana GLRs, AtGLR1.1 (AAF26802.1), AtGLR1.2 (BAA96960.1), AtGLR1.3 (BAA96961.2), AtGLR1.4 (AAF02156.1), AtGLR2.1 (AAB61068.1), AtGLR2.2 (AAD26895.1), AtGLR2.3 (AAD26894.1), AtGLR2.4 (CAA19752.1), AtGLR2.5 (CAB96656.1), AtGLR2.6 (CAB96653.1), AtGLR2.7 (AAC33239.1), AtGLR2.8 (AAC33237.1), AtGLR2.9 (AAC33236.1), AtGLR3.1 (AAF63223.1), AtGLR3.2 (CAA18740.1), AtGLR3.3 (AAG51316.1), AtGLR3.4 (AAB71458.1), AtGLR3.5 (AAC69939.1), AtGLR3.6 (CAB63012.1) and AtGLR3.7 (AAC69938.1). Residues important for ligand binding are highlighted in grey in domain one (a) and domain two (b).
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Figure 6. A multiple sequence alignment of the R. norvegicus GLR (AAA41240) with the A. thaliana GLRs, AtGLR2.9 (AAC33236.1) and AtGLR1.1 (AAF26802.1) shows locations of residues that have been implicated in ligand binding highlighted in grey.
The two regions that bind glutamate are shown. In domain one, relatively highly conserved residues at Pro478, Thr480 and Arg485 bind the backbone of glutamate (★). In domain two, residues corresponding to Ser654, Thr655 and Glu705 from the R. norvegicus GLR also bind to glutamate (◆).
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The multiscale docking algorithm runs a series of progressive iterations to explore the three-dimensional space of a protein molecule for the most likely binding sites of a chosen ligand. In early iterations, many possible binding sites are found, as all surfaces of the protein are available for ligand binding. As the algorithm proceeds, preferred binding sites are identified as they are ‘discovered’ iteration upon iteration. For those proteins where there is a good fit between the protein and the ligand, the iterations reveal that only one or several overlapping protein sites are optimal for that ligand.
Using this approach, the docking algorithm confirmed that glutamate bound to the R. norvegicus GLR at the site predicted by the crystal structure (Figures 7a–c and 8a,b). In contrast, attempts to dock glutamate to the AtGLR2.9 model failed. The final iteration revealed that there was a high likelihood that glutamate would only associate with the surface of the protein, and not in the predicted ligand-binding site (Figure 7d–f). Furthermore, attempts to ‘force’ glutamate into the predicted ligand-binding site in silico showed that this could not occur naturally because of significant steric hindrance (Figure 8c,d). This is attributable to the replacement of one of the key residues that are required for binding of the side chain carboxylate group of glutamate (Figure 8d). The residue in question, Thr655 of the R. norvegicus GLR, is conserved amongst all known mammalian GLRs (Figure 5). In AtGLR2.9, Thr655 is replaced by a bulky, hydrophobic Phe residue, which blocks the ligand-binding pocket where glutamate would dock (Figures 6 and 8d). In fact, Thr655 is replaced either by Phe or the bulky, hydrophobic branch-chain amino acids, Leu or Ile, in 18 of the 20 AtGLR subunits, and is missing altogether from one other (Figure 5). This suggests that glutamate is not the natural ligand for the majority of AtGLR subunits.
Figure 7. ‘Snapshots’ of the cumulative iterations from the multiscale docking algorithm, for the interactions of GLR subunits and putative ligands.
The full ligand-binding domain, showing the amino-acid residues important in binding in the ligand-binding pocket (in yellow), is shown with the potential binding sites (green dots).
(a) An early iteration of the R. norvegicus GLR docking with glutamate.
(b) A middle iteration of the R. norvegicus GLR docking with glutamate.
(c) The last cumulative iteration of the R. norvegicus GLR docking with glutamate. Note that all potential binding sites cluster in the ligand-binding pocket.
(d) An early iteration of AtGLR2.9 docking with glutamate.
(e) A middle iteration of AtGLR2.9 docking with glutamate.
(f) The last cumulative iteration of AtGLR2.9 docking with glutamate. Arrows denote the fact that no specific binding site has been identified.
(g) An early iteration of AtGLR2.9 docking with glycine.
(h) A middle iteration of AtGLR2.9 docking with glycine.
(i) The last cumulative iteration of AtGLR2.9 docking with glycine. Note that all potential binding sites coalesce to unify in a single site in the ligand-binding pocket.
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Figure 8. Molecular models of ligands binding to GLR subunits.
The full ligand-binding domains showing the amino-acid residues important in binding (in green) are shown with the ligands (in red) in panels (a,c,e,g,i), whereas the detailed ligand-binding pockets with the amino acids implicated in binding are shown with the ligands in panels (b,d,f,h,j).
(a) Model of the ligand-binding site of the R. norvegicus GLR docked with glutamate.
(b) Detailed model of the ligand-binding pocket of the R. norvegicus GLR docked with glutamate.
(c) Model of the ligand-binding site of AtGLR2.9 docked with glutamate, showing the regions of steric hindrance as coloured spheres.
(d) Detailed model of the ligand-binding pocket of AtGLR2.9 docked with glutamate, showing the regions of steric hindrance as coloured spheres.
(e) Model of the ligand-binding site of AtGLR2.9 binding to glycine.
(f) Detailed model of the ligand-binding pocket of AtGLR2.9 binding to glycine.
(g) Model of the ligand-binding site of AtGLR1.1 binding to glutamate.
(h) Detailed model of the ligand-binding pocket of AtGLR1.1 binding to glutamate.
(i) Model of the ligand-binding site of AtGLR2.9 binding to DNQX.
(j) Detailed model of the ligand-binding pocket of AtGLR2.9 binding to DNQX.
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There are two types of mammalian NMDA receptor subunits: glutamate and glycine receptors (Ivanovic et al., 1998). Glutamate is bound by NR2 subunits, whereas glycine is bound by NR1 and NR3 subunits (Ivanovic et al., 1998). All NR2 subunits possess a conserved glutamate-binding GST motif, corresponding to Gly653, Ser654 and Thr655 in the glutamate-binding site of the R. norvegicus GLR (Figure 5). In contrast, most of the AtGLR sequences are similar to the glycine-binding subunits NR1 and NR3, where Thr655 is replaced by amino acids with large, hydrophobic side chains (Figure 5).
To determine if glycine could function as a ligand for plant GLR subunits, glycine was docked to the AtGLR2.9 model. Glycine preferentially docks with the proposed glutamate-binding site (Figures 7g–i and 8e,f). The data strongly suggest that glycine is the natural agonist for the majority of AtGLRs. The one exception to this is AtGLR1.1, which contains the glutamate-binding GST motif in the correct region of domain 2 (Figures 5 and 6). When the ligand-binding domain of AtGLR1.1 was modelled on the R. norvegicus receptor, glutamate bound to the same residues as in the mammalian GLRs (Figure 8g,h). Thus, while the majority of AtGLR subunits should bind glycine, we predict that plant GLR channels may function with both glycine and glutamate. This prediction is entirely consistent with the observations that we made with respect to both the gating of [Ca2+]cyt and the changes in hypocotyl elongation.
Analysis of the modelling results highlights residues implicated in glutamate binding in mammalian GLRs that also appear important for glycine binding in the AtGLRs. For example, the Glu and Arg residues in the binding site appear to be important for ligand binding in both the R. norvegicus GLR (Glu705, Arg485) and the AtGLR (Glu724, Arg529). Both are perfectly conserved over all receptors investigated (Figure 5). Similarly, other amino acids implicated in establishing the three-dimensional structure of the ligand-binding region of domain 1, such as Thr480 and Ile481, are conserved between species (Figure 5). In contrast, the binding-site Pro residue at position 478 in the R. norvegicus GLR is replaced by Asp522 in AtGLR2.9, and appears to bind to the glycine via a side chain interaction. The replacement of Pro with Asp would anchor glycine more strongly within the binding site. In domain 2, Ser654 and Thr655 of the R. norvegicus GLR are replaced by non-polar Ala and Phe residues, respectively, in the AtGLR. The Ala681 residue in the AtGLR is predicted to bind glycine via its backbone nitrogen (much as Ser 654 binds glutamate in mammalian GLRs). Crucially, the Phe682 residue in AtGLR is not predicted to interact with glycine; rather, the presence of this amino acid hinders binding of other amino acids, such as glutamate, because of the presence of their side chains.
DNQX appears to have the capacity to bind to all plant GLR subunits. This finding supports the hypothesis that DNQX interacts with plant GLRs (Lam et al., 1998). Modelling with AtGLR2.9 showed that DNQX bound in the predicted ligand-binding pocket (Figure 8i,j). Thus, DNQX would be predicted to compete for the plant GLR ligand-binding pocket with other compounds, such as GLR agonists. This is entirely consistent with the predicted interaction of DNQX with animal GLRs. Importantly, these findings are also consistent with the observation that glutamate and glycine compete with DNQX in the gating of [Ca2+]cyt (Figure 2) and in the regulation of hypocotyl elongation (Figure 4), and substantiate the contention that glutamate and glycine mediate their effect at plant GLRs.
Future studies should aim to obtain empirical evidence for ligand binding to plant GLR subunits. The findings presented here suggest that this may not be a trivial task. Given the fact that the animal receptors function as heteromeric channels (Armstrong et al., 1998) and given the preponderance of plant genes encoding the different subunits, it may prove difficult to devise experimental systems that allow the re-constitution of the plant channels to provide an accurate indication of the in vivo ligands. Indeed, animal GLRs are unable to bind to their ligands when expressed as homomeric channels. However, the modelling work presented here should provide guidance for future studies aimed at determining the ligands for these receptors, at least in vitro. Where glycine might not have previously been considered as a ligand for these receptors, or where difficulties may have been encountered when using glutamate as a ligand, new alternatives can be considered. Furthermore, the contention that these receptors may not function as amino acid receptors, based on lack of glutamate binding (Davenport, 2002), may have to be re-assessed.