Ligand--protein interactions in the glutamate receptor

Fourier transform infrared spectroscopy was used to investigate ligand-protein interactions in the ligand-binding domain of the GluR4 glutamate receptor subunit. Glutamate binding induces more extensive secondary structural changes in the ligand-binding domain than does kainate binding. Glutamate also alters the hydrogen bonding strength of the single free cysteine side chain in the domain, while kainate does not. On the other hand, the interaction of a binding site arginine residue with kainate appears to be stronger than that with glutamate. These results identify chemical and structural differences that may explain the different functional characteristics of the two agonists acting on ionotropic glutamate receptors. In doing so, they complement and extend recent crystallographic structures of the ligand-binding domain.     

Towards a view of functioning dimeric metabotropic receptors

X-ray crystallography was used to solve the atomic structure of the ligand binding domain of the metabotropic glutamate receptor type1 homo-dimer, making it possible to show the conformational change of this domain upon glutamate binding. Studies of dimeric metabotropic receptors thereafter have focused on the respective roles and interaction of the two subunits, on the activation mechanisms following the structural rearrangements of the ligand-binding domain, and on the functional significance of polyvalent cations, the binding of which was identified in the crystal. The direct interaction between the GABA(B) receptor and the metabotropic glutamate receptor (mGluR1) has also attracted attention. Recently, attention has focused on incorporating these structural features into a functional view of the receptors.     

AMPA receptors and bacterial periplasmic amino acid-binding proteins share the ionic mechanism of ligand recognition.

In order to identify key structural determinants for ligand recognition, we subjected the ligand-binding domain of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-selective glutamate receptor GluR-D subunit to site-directed mutagenesis. Based on the analysis of the [3H]AMPA-binding properties of the mutated binding sites, we constructed a revised three-dimensional model of the ligand-binding site, different in many respects from previously published models. In particular, our results indicate that the residues Arg507 and Glu727 represent the structural and functional correlates of Arg77 and Asp161 in the homologous bacterial lysine/ornithine/arginine-binding protein and histidine-binding protein, and directly interact with the alpha-carboxyl and alpha-amino group of the bound ligand, respectively. In contrast, Glu424, implicated previously in ionic interactions with the alpha-amino group of the agonist, is unlikely to have such a role in ligand binding. Our results indicate that glutamate receptors share with the bacterial polar amino acid-binding proteins the fundamental mechanism of amino acid recognition.     

Chemistry and conformation of the ligand-binding domain of GluR2 subtype of glutamate receptors

In the present report, using vibrational spectroscopy we have probed the ligand-protein interactions for full agonists (glutamate and alpha-amino-5-methyl-3-hydroxy-4-isoxazole propionate (AMPA)) and a partial agonist (kainate) in the isolated ligand-binding domain of the GluR2 subunit of the glutamate receptor. These studies indicate differences in the strength of the interactions of the alpha-carboxylates for the various agonists, with kainate having the strongest interactions and glutamate having the weakest. Additionally, the interactions at the alpha-amine group of the agonists have also been probed by studying the environment of the non-disulfide-bonded Cys-425, which is in close proximity to the alpha-amine group. These investigations suggest that the interactions at the alpha-amine group are stronger for full agonists such as glutamate and AMPA as evidenced by the increase in the hydrogen bond strength at Cys-425. Partial agonists such as kainate do not change the environment of Cys-425 relative to the apo form, suggesting weak interactions at the alpha-amine group of kainate. In addition to probing the ligand environment, we have also investigated the changes in the secondary structure of the protein. Results clearly indicate that full agonists such as glutamate and AMPA induce similar secondary structural changes that are different from those of the partial agonist kainate; thus, a spectroscopic signature is provided for identifying the functional consequences of a specific ligand binding to this protein.     

Stereochemistry of quinoxaline antagonist binding to a glutamate receptor investigated by Fourier transform infrared spectroscopy

The stereochemistry of the interactions between quinoxaline antagonists and the ligand-binding domain of the glutamate receptor 4 (GluR4) have been investigated by probing their vibrational modes using Fourier transform infrared spectroscopy. In solution, the electron-withdrawing nitro groups of both compounds establish a resonance equilibrium that appears to stabilize the keto form of one of the cyclic amide carbonyl bonds. Changes in the 6,7-dinitro-2,3-dihydroxyquinoxaline vibrational spectra on binding to the glutamate receptor, interpreted within the framework of a published crystal structure, illuminate the stereochemistry of the interaction and suggest that the binding site imposes a more polarized electronic bonding configuration on this antagonist. Similar spectral changes are observed for 6-cyano-7-dinitro-2,3-dihydroxyquinoxaline, confirming that its interactions with the binding site are highly similar to those of 6,7-dinitro-2,3-dihydroxyquinoxaline and leading to a model of the 6-cyano-7-dinitro-2,3-dihydroxyquinoxaline-S1S2 complex, for which no crystal structure is available. Conformational changes within the GluR ligand binding domain were also monitored. Compared with the previously reported spectral changes seen on binding of the agonist glutamate, only a relatively small change is detected on antagonist binding. This correlation between the functional effects of different classes of ligand and the magnitude of the spectroscopic changes they induce suggests that the spectral data reflect physiologically relevant conformational processes.     

Evolution of glutamate interactions during binding to a glutamate receptor

Glutamate receptors are the predominant mediators of excitatory synaptic signals in the central nervous system and are important in learning and memory as well as in diverse neuropathologies including epilepsy and ischemia. Their primary function is to receive the chemical signal glutamate (1), which binds to an extracellular domain in the receptor, and convert it into an electrical signal through the formation of cation-permeable transmembrane channels. Recently described end-state apo and ligated structures of the ligand-binding domain of a rat glutamate receptor provide a first view of specific molecular interactions between the ligand and the receptor that are central to the allosteric regulation of function in this protein. Yet there is little information on the mechanism and the structures of intermediates (if any) formed during the ligand-binding process. Here we have used time-resolved vibrational spectroscopy to show that the process involves a sequence of interleaved ligand and protein changes that starts with the docking of glutamate at the alpha-carboxylate moiety and ends with the establishment of the interactions between the gamma-carboxylate of glutamate and the protein.     

Characterization of the functional role of the N-glycans in the AMPA receptor ligand-binding domain

The ligand-binding domains of AMPA receptor subunits carry two conserved N-glycosylation sites. In order to gain insight into the functional role of the corresponding N-glycans, we examined how the elimination of glycosylation at these sites (N407 and N414) affects the ligand-binding characteristics, structural stability, cell-surface expression, and channel properties of homomeric GluR-D (GluR4) receptor and its soluble ligand-binding domain (S1S2). GluR-D S1S2 protein expressed as a secreted protein in insect cells was found to be glycosylated at N407 and N414. No major differences in the ligand-binding properties were observed between the 'wild-type' S1S2 and non-glycosylated N407D/N414Q double mutant, or between S1S2 proteins expressed in the presence or absence of tunicamycin, an inhibitor of N-glycosylation. Purified glycosylated and non-glycosylated S1S2 proteins also showed similar thermostabilities as determined by CD spectroscopy. Full-length homomeric GluR-D receptor with N407D/N414Q mutation was expressed on the surface of HEK293 cells like the wild-type GluR-D. In outside-out patches, GluR-D and the N407D/N414Q mutant produced similar rapidly desensitizing current responses to glutamate and AMPA. We therefore report that the two conserved ligand-binding domain glycans do not play any major role in receptor-ligand interactions, do not impart a stabilizing effect on the ligand-binding domain, and are not critical for the formation and surface localization of homomeric GluR-D AMPA receptors in HEK293 cells.     

Determinants of antagonist binding at the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit, GluR-D. Role of the conserved arginine 507 and glutamate 727 residues.

Previous structural and mutagenesis studies indicate that the invariant alpha-amino and alpha-carboxyl groups of glutamate receptor agonists are engaged in polar interactions with oppositely charged, conserved arginine and glutamate residues in the ligand-binding domain of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor. To examine the role of these residues (R507 and E727 in the GluR-D subunit) in the discrimination between agonists and antagonists, we analyzed the ligand-binding properties of homomeric GluR-D and its soluble ligand-binding domain with mutations at these positions. Filter-binding assays using [3H]AMPA, an agonist, and [3H]Ro 48-8587, a high-affinity antagonist, as radioligands revealed that even a conservative mutation at R507 (R507K) resulted in the complete loss of both agonist and antagonist binding. In contrast, a negative charge at position 727 was necessary for agonist binding, whereas the isosteric mutation, E727Q, abolished all agonist binding but retained high-affinity binding for [3H]Ro 48-8587, displaceable by 7,8-dinitroquinoxaline-2,3-dione. Competition binding studies with antagonists representing different structural classes in combination with ligand docking experiments suggest that the role of E727 is antagonist-specific, ranging from no interaction to weak electrostatic interactions involving indirect and direct hydrogen bonding with the antagonist molecule. These results underline the importance of ion pair interaction with E727 for agonist activity and suggest that an interaction with R507, but not with E727, is essential for antagonist binding.     

A vibrational spectroscopic investigation of interactions of agonists with GluR0, a prokaryotic glutamate receptor

We have used Fourier transform infrared spectroscopy to provide a detailed picture of the interactions between the carboxylate groups of the ligands, glutamate, serine, and glutamine, with the ligand-binding domain of a prokaryotic ionotropic glutamate receptor (GluR0). The vibrational spectra indicate that the noncovalent interactions between the 1C(alpha)-carboxylate moiety of the ligand and the protein are stronger for glutamate than for serine and glutamine. These results correlate well with the higher affinity of glutamate for GluR0-S1S2 relative to the affinities of serine and glutamine. In addition, all three ligands induce similar changes in the vibrational spectra and intrinsic fluorescence of the protein, which indicates that all three ligands induce the same structural changes in the protein. These results are consistent with the recent crystal structures of the glutamate and serine bound forms of GluR0-S1S2 and in addition provide insights into the structure of the glutamine bound form of the protein.     

LRET investigations of conformational changes in the ligand binding domain of a functional AMPA receptor

The structural investigations using the soluble ligand binding domain of the AMPA subtype of the glutamate receptor have provided invaluable insight into the mechanistic pathway by which agonist binding to this extracellular domain mediates the formation of cation-selective channels in this protein. These structures, however, are in the absence of the transmembrane segments, the primary functional component of the protein. Here, we have used a modified luminescence resonance energy transfer based method to obtain distance changes due to agonist binding in the ligand binding domain in the presence of the transmembrane segments. These distance changes show that the cleft closure conformational change observed in the isolated ligand binding domain upon binding agonist is conserved in the receptor with the channel segments, thus establishing that the isolated ligand binding domain is a good model of the domain in the receptor containing the transmembrane segments.     

Stereochemistry of glutamate receptor agonist efficacy: engineering a dual-specificity AMPA/kainate receptor

Upon agonist binding, the bilobate ligand-binding domains of the ionotropic glutamate receptors (iGluR) undergo a cleft closure whose magnitude correlates broadly with the efficacy of the agonist. AMPA (alpha-amino-5-methyl-3-hydroxy-4-isoxazolepropionic acid) and kainate are nonphysiological agonists that distinguish between subsets of iGluR. Kainate acts with low efficacy at AMPA receptors. Here we report that the structure-based mutation L651V converts the GluR4 AMPA receptor into a dual-specificity AMPA/kainate receptor fully activated by both agonists. To probe the stereochemical basis of partial agonism, we have also investigated the correlation between agonist efficacy and a series of vibrational and fluorescence spectroscopic signals of agonist binding to the corresponding wild-type and mutant GluR4 ligand-binding domains. Two signals track the extent of channel activation: the maximal change in intrinsic tryptophan fluorescence and the environment of the single non-disulfide bonded C426, which appears to probe the strength of interactions with the ligand alpha-amino group. Both of these signals arise from functional groups that are poised to detect changes in the extent of channel cleft closure and thus provide additional information about the coupling between conformational changes in the ligand-binding domain and activation of the intact receptor.     

Picosecond dynamics of the glutamate receptor in response to agonist-induced vibrational excitation

Conformational changes of proteins are dominated by the excitation and relaxation processes of their vibrational states. To elucidate the mechanism of receptor activation, the conformation dynamics of receptors must be analyzed in response to agonist-induced vibrational excitation. In this study, we chose the bending vibrational mode of the guanidinium group of Arg485 of the glutamate receptor subunit GluR2 based on our previous studies, and we investigated picosecond dynamics of the glutamate receptor caused by the vibrational excitation of Arg485 via molecular dynamics simulations. The vibrational excitation energy in Arg485 in the ligand-binding site initially flowed into Lys730, and then into the J-helix at the subunit interface of the ligand-binding domain. Consequently, the atomic displacement in the subunit interface around an intersubunit hydrogen bond was evoked in about 3 ps. This atomic displacement may perturb the subunit packing of the receptor, triggering receptor activation.     

Human genetic polymorphisms in T1R1 and T1R3 taste receptor subunits affect their function

Umami is the typical taste induced by monosodium glutamate (MSG), which is thought to be detected by the heterodimeric G protein-coupled receptor, T1R1 and T1R3. Previously, we showed that MSG detection thresholds differ substantially between individuals and we further showed that nontaster and hypotaster subjects are associated with nonsynonymous single polymorphisms occurring in the T1R1 and T1R3 genes. Here, we show using functional expression that both amino acid substitutions (A110V and R507Q) in the N-terminal ligand-binding domain of T1R1 and the 2 other ones (F749S and R757C), located in the transmembrane domain of T1R3, severely impair in vitro T1R1/T1R3 response to MSG. A molecular model of the ligand-binding region of T1R1/T1R3 provides a mechanistic explanation supporting functional expression data. The data presented here support causal relations between the genotype and previous in vivo psychophysical studies in human evaluating sensitivity to MSG.     

Characterizing the energetic states of the GluR2 ligand binding domain core-dimer

Tetrameric ligand binding domains of the family of ionotropic glutamate receptors assemble as dimers-of-dimers. Crystallographic studies of several glutamate receptor subtype isolated core-dimers suggest a single stable dimeric conformation. A binding domain dimer has not been captured in other conformations without the aid of biochemical methods to disrupt a critical dimer interface. Molecular dynamics simulations and continuum electrostatics calculations reveal that the active glutamate bound form of the ligand-binding domain found in typical crystal structures is the preferred energetic state of the isolated core-dimer in the presence of agonist glutamate. A desensitized conformational state is a higher energy ligand-bound state of the core-dimer. The resting apo conformational state is comparatively the least energetically favored conformation and does not contain a single state but a set of energetically equivalent conformational core-dimer states. We hypothesize the energetic balance of an open versus closed transmembrane region must be included to characterize the absolute energetic states of the full receptor, which in the presence of the ligand is believed to be a desensitized state.     

Molecular evolution of glutamate receptors: a primitive signaling mechanism that existed before plants and animals diverged.

We performed a genealogical analysis of the ionotropic glutamate receptor (iGluR) gene family, which includes the animal iGluRs and the newly isolated glutamate receptor-like genes (GLR) of plants discovered in Arabidopsis. Distance measures firmly placed the plant GLR genes within the iGluR clade as opposed to other ion channel clades and indicated that iGluRs may be a primitive signaling mechanism that predated the divergence of animals and plants. Moreover, phylogenetic analyses using both parsimony and neighbor joining indicated that the divergence of animal iGluRs and plant GLR genes predated the divergence of iGluR subtypes (NMDA vs. AMPA/KA) in animals. By estimating the congruence of the various glutamate receptor gene regions, we showed that the different functional domains, including the two ligand-binding domains and the transmembrane regions, have coevolved, suggesting that they assembled together before plants and animals diverged. Based on residue conservation and divergence as well as positions of residues with respect to functional domains of iGluR proteins, we attempted to examine structure-function relationships. This analysis defined M3 as the most highly conserved transmembrane domain and identified potential functionally important conserved residues whose function can be examined in future studies.     

Molecular dynamics simulations of the ligand-binding domain of the ionotropic glutamate receptor GluR2.

Ionotropic glutamate receptors are essential for fast synaptic nerve transmission. Recent x-ray structures for the ligand-binding (S1S2) region of the GluR2 alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-sensitive receptor have suggested how differences in protein/ligand interactions may determine whether a ligand will behave as a full agonist. We have used multiple molecular dynamics simulations of 2-5 ns duration to explore the structural dynamics of GluR2 S1S2 in the presence and absence of glutamate and in a complex with kainate. Our studies indicate that not only is the degree of domain closure dependent upon interactions with the ligand, but also that protein/ligand interactions influence the motion of the S2 domain with respect to S1. Differences in domain mobility between the three states (apo-S1S2, glutamate-bound, and kainate-bound) are surprisingly clear-cut. We discuss how these changes in dynamics may provide an explanation relating the mechanism of transmission of the agonist-binding event to channel opening. We also show here how the glutamate may adopt an alternative mode of binding not seen in the x-ray structure, which involves a key threonine (T480) side chain flipping into a new conformation. This new conformation results in an altered pattern of hydrogen bonding at the agonist-binding site.     

To Gate or not to Gate: Are the Delta Subunits in the Glutamate Receptor Family Functional Ion Channels?

The two delta receptor subunits remain the most puzzling enigma within the ionotropic glutamate receptor family. Despite the recent elucidation of the ligand-binding domain structure of delta2, many fundamental questions with regard to the subunits’ mechanism of function still remain unanswered. Of necessity, the majority of studies on delta receptors focused on the metabotropic function of delta2, since electrophysiological approaches to date are limited to the characterization of spontaneous currents through the delta2-lurcher mutant. Indeed, accumulated evidence primarily from delta2-deficient transgenic mice suggest that major physiological roles of delta2 are mediated via metabotropic signaling by the subunit’s C terminus. Why then would the subunits retain a conserved ion channel domain if they do not form functional ion channels? Any progress with regard to ionotropic function of the two delta subunits has been hampered by their largely unknown pharmacology. Even now that a pharmacological profile for delta2 is being established on the basis of the ligand-binding domain structure, wild-type delta2 channels in heterologous expression systems stay closed in the presence of molecules that have been demonstrated to bind to the receptor’s ligand-binding domain. In this paper, we review the current knowledge of delta subunits focusing on the disputed ionotropic function.     

Constitutive activation of the N-methyl-D-aspartate receptor via cleft-spanning disulfide bonds

Although the N-methyl-D-aspartate (NMDA) receptor plays a critical role in the central nervous system, many questions remain regarding the relationship between its structure and functional properties. In particular, the involvement of ligand-binding domain closure in determining agonist efficacy, which has been reported in other glutamate receptor subtypes, remains unresolved. To address this question, we designed dual cysteine point mutations spanning the NR1 and NR2 ligand-binding clefts, aiming to stabilize these domains in closed cleft conformations. Two mutants, E522C/I691C in NR1 (EI) and K487C/N687C in NR2 (KN) were found to exhibit significant glycine- and glutamate-independent activation, respectively, and co-expression of the two subunits produced a constitutively active channel. However, both individual mutants could be activated above constitutive levels in a concentration-dependent manner, indicating that cleft closure does not completely prevent agonist association. Interestingly, whereas the NR2 KN disulfide was found to potentiate channel gating and M3 accessibility, NR1 EI exhibited the opposite phenotype, suggesting that the EI disulfide may trap the NR1 ligand-binding domain in a lower efficacy conformation. Furthermore, both mutants affected agonist sensitivity at the opposing subunit, suggesting that closed cleft stabilization may contribute to coupling between the subunits. These results support a correlation between cleft stability and receptor activation, providing compelling evidence for the Venus flytrap mechanism of glutamate receptor domain closure.     

A molecular envelope of the ligand-binding domain of a glutamate receptor in the presence and absence of agonist.

Solution scattering studies were performed on a ligand-binding domain (S1S2) of a glutamate receptor ion channel (GluR) in order to study GluR-binding and signal-transduction mechanisms. The core of the ligand-binding domain is homologous to prokaryotic periplasmic binding proteins (PBP), whose binding mechanism involves a dramatic cleft closure: the "Venus flytrap". Several models of GluR function have proposed that a similar cleft closure is induced by agonist binding. We have directly tested this putative functional homology by measuring the radius of gyration of S1S2 in the presence and absence of saturating concentrations of agonists. In contrast to the PBP, S1S2 shows no reduction in radius of gyration upon agonist binding, excluding a comparably large conformational change. Furthermore, we determined an ab initio molecular envelope for our S1S2 construct, which also contains the peptides that connect the PBP homology core to the three transmembrane domains and to an N-terminal domain. By fitting an atomic model of the ligand-binding domain core to the envelope of our extended construct, we were able to establish the likely position of these connecting peptides. Their positions relative to one another and to the expected sites of an agonist-induced conformational change suggest that ion channel gating and desensitization may involve more subtle and complex mechanisms than have been assumed based on the structural homology to the PBP.