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.     

Structure of the S1S2 glutamate binding domain of GLuR3

Glutamate receptors are the most prevalent excitatory neurotransmitter receptors in the vertebrate central nervous system. Determining the structural differences between the binding sites of different subtypes is crucial to our understanding of neuronal circuits and to the development of subtype specific drugs. The structures of the binding domain (S1S2) of the GluR3 (flip) AMPA receptor subunit bound to glutamate and AMPA and the GluR2 (flop) subunit bound to glutamate were determined by X‐ray crystallography to 1.9, 2.1, and 1.55 Å, respectively. Overall, the structure of GluR3 (flip) S1S2 is very similar to GluR2 (flop) S1S2 (backbone RMSD of 0.30 ± 0.05 for glutamate‐bound and 0.26 ± 0.01 for AMPA‐bound). The differences in the flip and flop isoforms are subtle and largely arise from one hydrogen bond across the dimer interface and associated water molecules. Comparison of the binding affinity for various agonists and partial agonists suggest that the S1S2 domains of GluR2 and GluR3 show only small differences in affinity, unlike what is found for the intact receptors (with the exception of one ligand, Cl‐HIBO, which has a 10‐fold difference in affinity for GluR2 vs. GluR3). Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Synthesis and in vitro pharmacology at AMPA and kainate preferring glutamate receptors of 4-heteroarylmethylidene glutamate analogues

2-Amino-3-[3-hydroxy-5-(2-thiazolyl)-4-isoxazolyl]propionic acid (1) is a potent AMPA receptor agonist with moderate affinity for native kainic acid (KA) receptors, whereas (S)-E-4-(2,2-dimethylpropylidene)glutamic acid (3) show high affinity for the GluR5 subtype of KA receptors and much lower affinity for the GluR2 subtype of AMPA receptors. As an attempt to develop new pharmacological tools for studies of GluR5 receptors, (S)-E-4-(2-thiazolylmethylene)glutamic acid (4a) was designed as a structural hybrid between 1 and 3. 4a was shown to be a potent GluR5 agonist and a high affinity ligand and to indiscriminately bind to the AMPA receptor subtypes GluR1-4 with lower affinities. Compounds 4b-h, in which the 2-thiazolyl substituent of 4a was replaced by other heterocyclic rings, which have previously been incorporated as 5-substituents in AMPA analogues, as exemplified by 1 were also synthesized. Compounds 4b-h were either inactive (4e,f) or weaker than 4a as affinity ligands for GluR1-4 and GluR5 with relative potencies comparable with those of the corresponding AMPA analogues as AMPA receptor agonists. Compounds 4a-h may be useful tools for the progressing pharmacophore mapping of the GluR5 agonist binding site.     

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.     

Crystal structures of the GluR5 and GluR6 ligand binding cores: molecular mechanisms underlying kainate receptor selectivity

Little is known about the molecular mechanisms underlying differences in the ligand binding properties of AMPA, kainate, and NMDA subtype glutamate receptors. Crystal structures of the GluR5 and GluR6 kainate receptor ligand binding cores in complexes with glutamate, 2S,4R-4-methylglutamate, kainate, and quisqualate have now been solved. The structures reveal that the ligand binding cavities are 40% (GluR5) and 16% (GluR6) larger than for GluR2. The binding of AMPA- and GluR5-selective agonists to GluR6 is prevented by steric occlusion, which also interferes with the high-affinity binding of 2S,4R-4-methylglutamate to AMPA receptors. Strikingly, the extent of domain closure produced by the GluR6 partial agonist kainate is only 3 degrees less than for glutamate and 11 degrees greater than for the GluR2 kainate complex. This, together with extensive interdomain contacts between domains 1 and 2 of GluR5 and GluR6, absent from AMPA receptors, likely contributes to the high stability of GluR5 and GluR6 kainate complexes.     

Molecular dynamics simulations of the conformational changes of the glutamate receptor ligand-binding core in the presence of glutamate and kainate

Excitatory synaptic transmission is mediated by ionotropic glutamate receptors (iGluRs) through the induced transient opening of transmembrane ion channels. The three-dimensional structure of the extracellular ligand-binding core of iGluRs shares the overall features of bacterial periplasmic binding proteins (PBPs). In both families of proteins, the ligand-binding site is arranged in two domains separated by a cleft and connected by two peptide stretches. PBPs undergo a typical hinge motion of the two domains associated with ligand binding that leads to a conformational change from an open to a closed form. The common architecture suggests a similar closing mechanism in the ligand-binding core of iGluRs induced by the binding of specific agonists. Starting from the experimentally determined kainate-bound closed form of the S1S2 GluR2 construct, we have studied by means of molecular dynamics simulations the opening motion of the ligand-binding core in the presence and in the absence of both glutamate and kainate. Our results suggest that the opening/closing interdomain hinge motions are coupled to conformational changes in the insertion region of the transmembrane segments. These changes are triggered by the interaction of the agonists with the essential Glu 209 residue. A plausible mechanism for the coupling of agonist binding to channel gating is discussed.     

Mechanism of activation and selectivity in a ligand-gated ion channel: structural and functional studies of GluR2 and quisqualate

Glutamate is the major excitatory neurotransmitter in the mammalian brain. The (S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazole)propionic acid (AMPA)-subtype glutamate receptor, a ligand-gated ion channel, mediates most of the fast excitatory synaptic transmission in the mammalian central nervous system. Here we present electrophysiological, biochemical, and crystallographic data on the interactions between quisqualate and the GluR2 receptor ion channel and its corresponding ligand binding core. Quisqualate is a high-affinity, full agonist which like AMPA and glutamate elicits maximum peak current responses, and stabilizes the ligand binding core in a fully closed conformation, reinforcing the concept that full agonists produce similar conformational changes [Armstrong, N., and Gouaux, E. (2000) Neuron 28, 165-181]. Nevertheless, the mechanism of quisqualate binding is different from that of AMPA but similar to that of glutamate, illustrating that quisqualate is a faithful glutamate analogue. A detailed comparison of the three agonist complexes reveals distinct binding mechanisms, particularly in the region of a hydrophobic pocket that is proximal to the anionic gamma-substituents, and demonstrates the importance of agonist-water-receptor interactions. The hydrophobic pocket, which is predicted to vary in chemical character between receptor subtypes, probably plays an important role in determining receptor subtype specificity.     

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.     

Probing the function,conformational plasticity,and dimer-dimer contacts of the GluR2 ligand-binding core: studies of 5-substituted willardiines and GluR2 S1S2 in the crystal

Numerous naturally occurring and synthetic alpha-amino acids act as agonists on (S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazole) propionic acid (AMPA) receptors but nevertheless display significant differences in their functional properties and modes of interaction. The 5-substituted willardiines are a series of compounds that exhibit a range of affinities, act as partial agonists, and give rise to intermediate levels of activation and desensitization. However, the molecular basis for the activities of 5-substituted willardiines has not been conclusively elaborated at the level of atomic resolution. Here we provide insight into the molecular basis of the potency and efficacy elicited by the 5-substituted willardiines on the basis of cocrystal structures with the GluR2 ligand-binding core. We also show that the crystallized ligand-binding core has an affinity for agonists similar to the ligand-binding core in solution. Analysis of multiple crystal lattices suggests modes by which the ligand-binding core dimers interact in the tetrameric receptor. These studies further our understanding of how subtle differences in the structures of agonists are correlated to changes in the conformation of residues and water molecules in the immediate binding pocket and to the degree of domain closure.     

Tetrazolyl isoxazole amino acids as ionotropic glutamate receptor antagonists: synthesis, modelling and molecular pharmacology

Two 3-(5-tetrazolylmethoxy) analogues, 1a and 1b, of (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA), a selective AMPA receptor agonist, and (RS)-2-amino-3-(5-tert-butyl-3-hydroxy-4-isoxazolyl)propionic acid (ATPA), a GluR5-preferring agonist, were synthesized. Compounds 1a and 1b were pharmacologically characterized in receptor binding assays, and electrophysiologically on homomeric AMPA receptors (GluR1-4), homomeric (GluR5 and GluR6) and heteromeric (GluR6/KA2) kainic acid receptors, using two-electrode voltage-clamped Xenopus laevis oocytes expressing these receptors. Both analogues proved to be antagonists at all AMPA receptor subtypes, showing potencies (Kb=38-161 microM) similar to that of the AMPA receptor antagonist (RS)-2-amino-3-[3-(carboxymethoxy)-5-methyl-4-isoxazolyl]propionic acid (AMOA) (Kb=43-76 microM). Furthermore, the AMOA analogue, 1a, blocked two kainic acid receptor subtypes (GluR5 and GluR6/KA2), showing sevenfold preference for GluR6/KA2 (Kb=19 microM). Unlike the iGluR antagonist (S)-2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl]propionic acid [(S)-ATPO], the corresponding tetrazolyl analogue, 1b, lacks kainic acid receptor effects. On the basis of docking to a crystal structure of the isolated extracellular ligand-binding core of the AMPA receptor subunit GluR2 and a homology model of the kainic acid receptor subunit GluR5, we were able to rationalize the observed structure-activity relationships.     

Discrimination between agonists and antagonists by the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid-selective glutamate receptor. A mutation analysis of the ligand-binding domain of GluR-D subunit

The crystal structures of the ligand-binding core of the agonist complexes of the glutamate receptor-B (GluR-B) subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-selective glutamate receptor indicate that the distal anionic group of agonist molecules are stabilized by interactions with an N-terminal region of an alpha-helix (helix F) in the lobe 2 ("domain 2," Armstrong, N., and Gouaux, E. (2000) Neuron 28, 165-181) of the two-lobed ligand-binding domain. We used site-directed mutagenesis to further analyze the role of this region in the recognition of both agonists and antagonists by the AMPA receptor. Wild-type and mutated versions of the ligand-binding domain of GluR-D were expressed in insect cells as secreted soluble polypeptides and subjected to binding assays using [(3)H]AMPA, an agonist, and [(3)H]Ro 48-8587 (9-imidazol-1-yl-8-nitro-2,3,5,6-tetrahydro[1,2,4]triazolo[1,5-c] quinazoline-2,5-dione), a high affinity AMPA receptor antagonist, as radioligands. Single alanine substitutions at residues Leu-672 and Thr-677 severely affected the affinities for all agonists, as seen in ligand competition assays, whereas similar mutations at residues Asp-673, Ser-674, Gly-675, Ser-676, and Lys-678 selectively affected the binding affinities of one or two of the agonists. In striking contrast, the binding affinities of [(3)H]Ro 48-8587 and of another competitive antagonist, 6,7-dinitroquinoxaline-2,3-dione, were not affected by any of these alanine mutations, suggesting the absence of critical side-chain interactions. Together with ligand docking experiments, our results indicate a selective engagement of the side chains of the helix F region in agonist binding, and suggest that conformational changes involving this region may play a critical role in receptor activation.     

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.     

Differential effects of chronic hyperammonemia on modulation of the glutamate-nitric oxide-cGMP pathway by metabotropic glutamate receptor 5 and low and high affinity AMPA receptors in cerebellum in vivo

Previous studies show that chronic hyperammonemia impairs learning ability of rats by impairing the glutamate-nitric oxide (NO)-cyclic guanosine mono-phosphate (cGMP) pathway in cerebellum. Three types of glutamate receptors cooperate in modulating the NO-cGMP pathway: metabotropic glutamate receptor 5 (mGluR5), (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-d-aspartic acid (NMDA) receptors. The aim of this work was to assess whether hyperammonemia alters the modulation of this pathway by mGluR5 and AMPA receptors in cerebellum in vivo. The results support that in control rats: (1) low AMPA concentrations (0.1mM) activate nearly completely Ca(2+)-permeable (glutamate receptor subunit 2 (GluR2)-lacking) AMPA receptors and the NO-cGMP pathway; (2) higher AMPA concentrations (0.3 mM) also activate Ca(2+)-impermeable (GluR2-containing) AMPA receptors, leading to activation of NMDA receptors and of NO-cGMP pathway. Moreover, the data support that chronic hyperammonemia: (1) reduces glutamate release and activation of the glutamate-NO-cGMP pathway by activation of mGluR5; (2) strongly reduces the direct activation by AMPA receptors of the NO-cGMP pathway, likely due to reduced entry of Ca(2+) through GluR2-lacking, high affinity AMPA receptors; (3) strongly increases the indirect activation of the NO-cGMP pathway by high affinity AMPA receptors, likely due to increased entry of Na(+) through GluR2-lacking AMPA receptors and NMDA receptors activation; (4) reduces the indirect activation of the NO-cGMP pathway by low affinity AMPA receptors, likely due to reduced activation of NMDA receptors.     

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.     

Molecular mechanism of agonist recognition by the ligand-binding core of the ionotropic glutamate receptor 4

The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) class of ionotropic glutamate receptors comprises four different subunits: iGluR1/iGluR2 and iGluR3/iGluR4 forming two subgroups. Three-dimensional structures have been reported only of the ligand-binding core of iGluR2. Here, we present two X-ray structures of a soluble construct of the R/G unedited flip splice variant of the ligand-binding core of iGluR4 (iGluR4_i(R)-S1S2) in complex with glutamate or AMPA. Subtle, but important differences are found in the ligand-binding cavity between the two AMPA receptor subgroups at position 724 (Tyr in iGluR1/iGluR2 and Phe in iGluR3/iGluR4), which in iGluR4 may lead to displacement of a water molecule and hence points to the possibility to make subgroup specific ligands.     

Structural basis for AMPA receptor activation and ligand selectivity: crystal structures of five agonist complexes with the GluR2 ligand-binding core

Glutamate is the principal excitatory neurotransmitter within the mammalian CNS, playing an important role in many different functions in the brain such as learning and memory. In this study, a combination of molecular biology, X-ray structure determinations, as well as electrophysiology and binding experiments, has been used to increase our knowledge concerning the ionotropic glutamate receptor GluR2 at the molecular level. Five high-resolution X-ray structures of the ligand-binding domain of GluR2 (S1S2J) complexed with the three agonists (S)-2-amino-3-[3-hydroxy-5-(2-methyl-2H-tetrazol-5-yl)isoxazol-4-yl]propionic acid (2-Me-Tet-AMPA), (S)-2-amino-3-(3-carboxy-5-methylisoxazol-4-yl)propionic acid (ACPA), and (S)-2-amino-3-(4-bromo-3-hydroxy-isoxazol-5-yl)propionic acid (Br-HIBO), as well as of a mutant thereof (S1S2J-Y702F) in complex with ACPA and Br-HIBO, have been determined. The structures reveal that AMPA agonists with an isoxazole moiety adopt different binding modes in the receptor, dependent on the substituents of the isoxazole. Br-HIBO displays selectivity among different AMPA receptor subunits, and the design and structure determination of the S1S2J-Y702F mutant in complex with Br-HIBO and ACPA have allowed us to explain the molecular mechanism behind this selectivity and to identify key residues for ligand recognition. The agonists induce the same degree of domain closure as AMPA, except for Br-HIBO, which shows a slightly lower degree of domain closure. An excellent correlation between domain closure and efficacy has been obtained from electrophysiology experiments undertaken on non-desensitising GluR2i(Q)-L483Y receptors expressed in oocytes, providing strong evidence that receptor activation occurs as a result of domain closure. The structural results, combined with the functional studies on the full-length receptor, form a powerful platform for the design of new selective agonists.     

Functional expression from cloned cDNAs of glutamate receptor species responsive to kainate and quisqualate.

The complete amino acid sequences of two mouse glutamate receptor subunits (GluR1 and GluR2) have been deduced by cloning and sequencing the cDNAs. Xenopus oocytes injected with mRNA derived from the GluR1 cDNA exhibit current responses both to kainate and to quisqualate as well as to glutamate, whereas oocytes injected with mRNA derived from the GluR2 cDNA show little response. Injection of oocytes with both the mRNAs produces current responses larger than those induced by the GluR1-specific mRNA and the dose-response relations indicate a positively cooperative interaction between the two subunits. These results suggest that kainate and quisqualate can activate a common glutamate receptor subtype and that glutamate-gated ionic channels are hetero-oligomers of different subunits.     

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.     

Mechanisms for activation and antagonism of an AMPA-sensitive glutamate receptor: crystal structures of the GluR2 ligand binding core

Crystal structures of the GluR2 ligand binding core (S1S2) have been determined in the apo state and in the presence of the antagonist DNQX, the partial agonist kainate, and the full agonists AMPA and glutamate. The domains of the S1S2 ligand binding core are expanded in the apo state and contract upon ligand binding with the extent of domain separation decreasing in the order of apo > DNQX > kainate > glutamate approximately equal to AMPA. These results suggest that agonist-induced domain closure gates the transmembrane channel and the extent of receptor activation depends upon the degree of domain closure. AMPA and glutamate also promote a 180 degrees flip of a trans peptide bond in the ligand binding site. The crystal packing of the ligand binding cores suggests modes for subunit-subunit contact in the intact receptor and mechanisms by which allosteric effectors modulate receptor activity.