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J. Biol. Chem., Vol. 282, Issue 35, 25726-25736, August 31, 2007

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Partial Agonism and Antagonism of the Ionotropic Glutamate Receptor iGLuR5

STRUCTURES OF THE LIGAND-BINDING CORE IN COMPLEX WITH DOMOIC ACID AND 2-AMINO-3-[5-tert-BUTYL-3-(PHOSPHONOMETHOXY)-4-ISOXAZOLYL]PROPIONIC ACID^*

Helle Hald¹, Peter Naur¹, Darryl S. Pickering^¶, Desiree Sprogøe, Ulf Madsen, Daniel B. Timmermann, Philip K. Ahring, Tommy Liljefors, Arne Schousboe^¶, Jan Egebjerg^||, Michael Gajhede, and Jette Sandholm Kastrup²

From the Departments of Medicinal Chemistry and ^¶Pharmacology and Pharmacotherapy, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100, Copenhagen, the NeuroSearch A/S, Pederstrupvej 93, DK-2750 Ballerup, and the ^||H. Lundbeck A/S, Ottiliavej 9, DK-2500 Valby, Denmark

Received for publication, January 5, 2007 , and in revised form, April 19, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
More than 50 structures have been reported on the ligand-binding core of the ionotropic glutamate receptor iGluR2 that belongs to the 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid-type of receptors. In contrast, the ligand-binding core of the kainic acid-type receptor iGluR5 has only been crystallized with three different ligands. Hence, additional structures of iGluR5 are needed to broaden the understanding of the ligand-binding properties of iGluR5, and the conformational changes leading to channel opening and closing. Here, we present two structures of the ligand-binding core of iGluR5; one as a complex with the partial agonist (2S,3S,4S)-3-carboxymethyl-4-[(1Z,3E,5R)-5-carboxy-1-methyl-hexa-1,3-dienyl]-pyrrolidine-2-carboxylic acid (domoic acid) and one as a complex with the antagonist (S)-2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl]propionic acid ((S)-ATPO). In agreement with the partial agonist activity of domoic acid, the ligand-binding core of the iGluR5 complex is stabilized by domoic acid in a conformation that is 11° more open than the conformation observed in the full agonist (S)-glutamic acid complex. This is primarily caused by the 5-carboxy-1-methyl-hexa-1,3-dienyl moiety of domoic acid and residues Val⁶⁸⁵-Thr⁶⁹⁰ of iGluR5. An even larger domain opening of 28° is introduced upon binding of the antagonist (S)-ATPO. It appears that the span of domain opening is much larger in the ligand-binding core of iGluR5 (30°) compared with what has been observed in iGluR2 (19°). Similarly, much larger variation in the distances between transmembrane linker residues in the two protomers comprising the dimer is observed in iGluR5 as compared with iGluR2.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
The ionotropic glutamate receptors (iGluRs)³ are responsible for the major part of the fast excitatory synaptic transmission in the mammalian brain. The iGluRs are divided into N-methyl-D-aspartic acid, 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA), and kainic acid receptors based on selective agonist binding properties and sequence similarity of the receptor subunits (1-3). The three classes of iGluRs each consist of a number of subunits: NR1, NR2A-D, and NR3A-B for N-methyl-D-aspartic acid receptors, iGluR1-4 for AMPA receptors, and iGluR5-7 and KA1-2 for kainic acid receptors. The iGluRs share a similar tetrameric structure where the subunits assemble as a set of two dimers (4-8). Each subunit contains a bi-lobed ligand-binding core attached to the transmembrane regions forming the ion channel pore. After the first crystal structure of the genetically engineered soluble form of the iGluR2 ligand-binding core (iGluR2-S1S2J) was reported, representative structures have been published for members of all three iGluR classes (9-13). The majority of the structures, however, are co-crystals of iGluR2-S1S2J and agonists, which in combination with biophysical and biochemical analysis have provided molecular details of the receptor activation mechanisms. Surprisingly, only a few crystal structures have been solved of antagonist complexes (9, 12, 14-16).

The structure of the ligand-binding core of the kainic acid receptor subunit iGluR5 (iGluR5-S1S2) has so far only been reported in complex with the endogenous ligand (S)-glutamic acid (10, 11, 16), and recently with two structurally related antagonists (S)-1-(2-amino-2-carboxyethyl)-3-(2-carboxybenzyl)pyrimidine-2,4-dione (UBP302) and (S)-1-(2-amino-2-carboxyethyl)-3-(2-carboxythiophene-3-yl-methyl)-5-methylpyrimidine-2,4-dione (UBP310) (16) (see Fig. 1). Here, a number of differences were observed between the iGluR2 and the iGluR5 structures. Despite a tighter domain closure induced by (S)-glutamic acid in iGluR5 compared with iGluR2, the differences in amino acid residues in the binding pocket generated a 40% larger cavity in iGluR5-S1S2 (11). Furthermore, a highly coordinated network of water molecules interacting with Ser⁷⁴¹ stabilizes an inter-domain bridge in the iGluR5-S1S2 complex not seen in iGluR2-S1S2J (10).

View larger version (14K): [in this window] [in a new window]   FIGURE 1. Chemical structures of domoic acid, kainic acid, (S)-ATPO, UBP302, and UBP310. The atom numbering of domoic acid is shown in italics and is according to Nanao et al. (46) (PDB entry code 1YAE). The numbering of (S)-ATPO is according to Hogner et al. (14) (PDB entry code 1N0T). See abbreviations for compound names.

A number of iGluR agonists and antagonists have been developed by use of the isoxazole nucleus as a scaffold (3, 29). The compound 2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl]propionic acid (ATPO; Fig. 1) was developed as a hybrid structure based on the iGluR5 agonist 2-amino-3-(5-tert-butyl-3-hydroxy-4-isoxazolyl)propionic acid and long-chain phosphonic acid containing N-methyl-D-aspartic acid receptor antagonists such as 2-amino-7-phosphonoheptanoic acid. ATPO was first described in its racemic form as a competitive AMPA receptor antagonist (30). Upon resolution of the ATPO enantiomers, a more detailed study was performed of the pharmacology on recombinant AMPA and kainic acid receptors expressed in Xenopus laevis oocytes (31). In these studies, (S)-ATPO was shown to be an antagonist at iGluR1-4 (K_i values ranging from 2.0 to 6.7 µM) and less potent at iGluR5 (K_i = 23 µM), but no activity was observed at iGluR6 or KA2. The R-enantiomer was virtually inactive at all subtypes (31). Important information on the binding mode of ATPO was obtained by determination of a crystal structure of (S)-ATPO in complex with the iGluR2 ligand-binding core (14). This study provided detailed information about iGluR antagonism and in particular showed how (S)-ATPO stabilizes an open form of the ligand-binding core.

In the present study, domoic acid and (S)-ATPO were selected as obvious ligands to gain further insight into the mechanism of kainic acid receptor versus AMPA receptor agonism and antagonism. At the same time, information on the mechanism of selectivity within the kainic acid receptor class was revealed. Thus, we present x-ray crystallographic data of iGluR5-S1S2 in complex with domoic acid and (S)-ATPO. These two structures, combined with the previously reported structures of iGluR5-S1S2 in complex with (S)-glutamic acid (10, 11, 16) and in complex with the antagonists UBP302 and UBP310 (16) allow a comparison of iGluR5 ligand-binding core domain opening and distances between the transmembrane TM1-TM2 linker amino acids of each protomer comprising the crystallographic dimer.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Materials—Chemicals were purchased from Sigma, unless otherwise specified. Domoic acid was obtained from Tocris Cookson (United Kingdom). (S)-ATPO was synthesized, purified, and resolved as previously described (30, 31). The rat iGluR5-S1S2 construct was expressed and purified as previously reported (10).

Radioligand Binding Assay—For saturation binding, 50 ng of iGluR5-S1S2 protein was incubated with 0.065-18 nM (2S,4R)-[³H]4-methylglutamic acid (47.9 Ci/mmol, Tocris Cookson, UK) for 2 h on ice in assay buffer (50 mM Tris-HCl, 10% (v/v) glycerol, pH 7.1, at 4 °C). Nonspecific binding was determined in the presence of 1 mM (S)-glutamic acid. Competition experiments were carried out using 35 ng of iGluR5-S1S2 with 0.5-2 nM radiolabel in the presence of 0.01-1,000 nM domoic acid or 0.10-500 µM (R,S)-ATPO. Samples were filtered onto Schleicher & Schuell 0.2-µm ME24 mixed cellulose ester filters and washed twice with 2 ml of ice-cold assay buffer. Radioligand binding to full-length iGluR5(Q) expressed in Sf9 insect cells was carried out as previously detailed (32). Radioactivity was determined by scintillation counting. Data were analyzed using Grafit version 3.00 (Erithacus Software Ltd., Horley, UK) and fit as previously described (Ref. 33, Equations 3 and 4) to determine the Hill coefficient and K_i. Saturation binding data were fit as total bound to an equation accounting for ligand depletion at a single binding site (Ref. 33, Equation 2), with the nonspecific binding fit as a linear component of total binding.

Crystallization—A solution of 2.3 mg/ml iGluR5-S1S2 (in 10 mM HEPES, pH 7.0, 20 mM NaCl, and 1 mM EDTA) containing 5mM domoic acid was used for the co-crystallization experiments, giving a protein to ligand ratio of 1:50. Crystallization was carried out at 6 °C using the hanging drop vapor diffusion method. The drops contained 1 µl of protein-ligand solution and 1 µl of reservoir solution; the reservoir solution contained 15% polyethylene glycol 4000, 0.3 M lithium sulfate, and 0.1 M cacodylate, pH 6.5.

For co-crystallization with (S)-ATPO, a 6 mg/ml iGluR5-S1S2 solution (in 10 mM HEPES, pH 7.0, 20 mM NaCl, and 1 mM EDTA) was diluted 1:1 with ligand solution (10 mM HEPES, pH 7.0, 20 mM NaCl, 1 mM EDTA, and 40 mM (S)-ATPO) and used for crystallization experiments. Crystals were obtained at 6 °C and at several conditions that all included polyethylene glycol 4000 and 0.3 M lithium sulfate but with buffers at different pH values. The crystal used for data collection was grown from 20% polyethylene glycol 4000, 0.3 M lithium sulfate, and 0.1 M cacodylate, pH 6.5.

Data Collections and Processing—Crystals of iGluR5-S1S2: domoic acid were flash cooled in liquid nitrogen after soaking in a cryo-protectant containing 20% glycerol in reservoir solution. X-ray diffraction data were collected at the BL2 beamline at BESSY, Berlin, Germany, equipped with a MAR345 image plate detector. The wavelength was 0.954 Å and the crystal diffracted to 2.5 Å. Data processing was performed using the HKL programs Denzo, XdisplayF, and Scalepack (34), and the CCP4 suite of programs (35).

View larger version (49K): [in this window] [in a new window]   FIGURE 2. Schematic structure of the ligand-binding core of iGluR5 in complex with domoic acid. A 2-fold symmetric dimer is present in the crystal (molA is shown in blue and molB in green). Domoic acid is displayed in orange. The distance between two Ile668 (shown as spheres) of the iGluR5 dimer is indicated by a double arrow, representing the TM1-TM2 connection to the transmembrane region.

View larger version (38K): [in this window] [in a new window]   FIGURE 3. Binding mode of the agonist domoic acid in iGluR5-S1S2. A, Fo - Fc omit electron-density map of domoic acid contoured at 3. Oxygen atoms are colored red, nitrogen atoms are blue, and carbon atoms are yellow. The chemical structure of domoic acid is shown in Fig. 1. B, close-up view of the iGluR5-S1S2·domoic acid complex (molB) including potential hydrogen bonds within 3.0 Å (dotted lines). Bonds of protein and ligand are shown in green and yellow, respectively. Water molecules are shown as red spheres. C, close-up view of the selectivity determining loops of iGluR2 (red), iGluR5 (green), and iGluR6 (blue). This loop corresponds to the region around Arg686 and Asp687 of domain 2 in iGluR5. The structures of iGluR6-S1S2:domoic acid (PDB code 1YAE) (46) and iGluR2-S1S2J:kainic acid (PDB code 1FW0) (9) were superimposed onto the iGluR5-S1S2: domoic acid structure (domain 1 residues). In iGluR5 and iGluR6, domoic acid is colored green and blue, respectively. Kainic acid in iGluR2 is colored red. The chemical structure of kainic acid is shown in Fig. 1.

The structure of iGluR5-S1S2 in complex with (S)-ATPO was solved by molecular replacement using the program Phaser (40) with the structure of iGluR2-S1S2J in complex with (S)-ATPO (molA, PDB entry code 1N0T) (14) as search model. The program ArpWarp (41) was used to trace 96% of the residues. Further model building was done using the program Coot (37) cycled with refinement using the CNS package (38). The fully refined structure comprises Thr⁴³³-Gln⁴⁹², Trp⁴⁹⁸-Lys⁵⁴⁴, the Gly-Thr linker, Pro⁶⁶⁷-Ser⁷¹¹, and Ser⁷¹⁵-Gly⁸⁰³. For statistics on refinements, see Table 1.

Structure Analysis and Figure Preparation—The contacts program within CCP4 (35) was used to analyze the structures (hydrogen bonds and van der Waals interactions). Domain openings were calculated using DynDom (42). The dimerization interfaces were analyzed using the Protein-Protein Interaction Server (43). Figs. 2, 3, 5, and 6 were prepared with PyMol (44) and Fig. 4 with MOE (45).

Protein Data Bank Accession Numbers—The atomic coordinates and structure factor amplitudes of the iGluR5-S1S2:domoic acid and iGluR5-S1S2:(S)-ATPO structures have been deposited in the RCSB Protein Data Bank under accession numbers 2PBW and 1VSO.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
The ligand-binding core of iGluR5 was crystallized in complex with the agonist domoic acid and the antagonist (S)-ATPO, and the structures were determined to 2.5- and 1.85-Å resolution, respectively. The binding affinities of these two ligands (K_i) for the iGluR5-S1S2 construct were determined by competition studies with [³H]-(2S,4R) 4-methylglutamic acid (Table 2). The K_i values of domoic acid at iGluR5-S1S2 (5.56 nM) and at the full-length iGluR5(Q) receptor (1.11 nM) were found to be similar. The binding affinity of ATPO at iGluR5-S1S2 (19.0 µM) seems to be lower than at the full-length iGluR5(Q) receptor (2.21 µM) in this assay, but is in agreement with previous studies (23 µM) (31). Hill values of unity for all compounds indicate one homogeneous binding site population for both full-length iGluR5(Q) and iGluR5-S1S2.

The Structure of iGluR5-S1S2 in Complex with Domoic Acid—In the crystal structure of iGluR5-S1S2 in complex with domoic acid, two molecules are present in the asymmetric unit of the crystal (molA and molB; Fig. 2). The electron density of domoic acid in the ligand-binding site of iGluR5-S1S2 is well defined and allowed unambiguous positioning of the ligand and identification of ligand contacts; see Fig. 3A. Domoic acid interacts with residues of iGluR5-S1S2 via hydrogen bonds, ionic interactions, van der Waals interactions, and water-mediated contacts. The potential hydrogen bonds/ionic interactions are listed in Table 3 and illustrated in Figs. 3B and 4A. The -amino acid part of domoic acid interacts with residues Pro⁵¹⁶, Thr⁵¹⁸, Arg⁵²³, Ser⁶⁸⁹, and Glu⁷³⁸. Ser⁶⁸⁹, together with Thr⁶⁹⁰, is also involved in interactions with the acetic acid part of domoic acid. Furthermore, the carboxylate group on the hexa-1,3-dienyl moiety of domoic acid forms a hydrogen bond to the backbone nitrogen atom of Tyr⁴⁸⁹. A number of water-mediated contacts are seen between iGluR5-S1S2 and domoic acid. The acetic acid carboxylate group of domoic acid makes indirect contacts to Thr⁶⁹⁰, Val⁶⁸⁵, Met⁶⁹¹, Leu⁷³⁶, and Ser⁷⁴¹ through three water molecules (Table 3). The previously shown water-mediated network (via three water molecules) from Ser⁷⁴¹ to (S)-glutamic acid observed in the iGluR5-S1S2:(S)-glutamic acid complex (10) is only partly conserved in the present domoic acid complex, because the water molecule interacting with the nitrogen atom of glutamic acid is displaced by the pyrrolidine ring of domoic acid (not shown).

View larger version (23K): [in this window] [in a new window]   FIGURE 4. Two-dimensional ligand-receptor interaction plots of iGluR5-S1S2 in complex with domoic acid (A) and (S)-ATPO (B). iGluR5-S1S2 polar residues are shown as purple circles, acidic residues as purple/red, basic residues as purple/blue, and hydrophobic residues in green. Contacts from ligand to receptor side chains as calculated by the program MOE are shown as green arrows and from ligand to receptor backbone as blue arrows. Water molecules in contact with ligands are shown as white circles and their contacts as yellow lines. The extent of ligand and receptor exposure is shown as blue spheres differing in size.

The Structure of iGluR5-S1S2 Complexed with (S)-ATPO—The crystals of iGluR5-S1S2 in complex with the antagonist (S)-ATPO contain one molecule within the asymmetric unit of the crystal. The electron density of (S)-ATPO is very well defined; see Fig. 5A. The amino acid moiety of (S)-ATPO is anchored primarily by a bidentate salt bridge between the carboxylate group and Arg⁵²³ as well as by hydrogen bonds between the ammonium group and residues Pro⁵¹⁶, Thr⁵¹⁸, and Glu⁷³⁸ (Figs. 4B and 5B; Table 3); a motif that is common for binding of iGluR ligands. The phosphonate group of (S)-ATPO is anchored to domain 2 by Ser⁶⁸⁹ only; however, water-mediated contacts to Thr⁶⁹⁰ and contact to a glycerol molecule are also observed.

In iGluR5-S1S2, the network of water molecules that connects Ser⁷⁴¹ to both the amino group and the distal carboxylic acid of (S)-glutamic acid through hydrogen bonds (10) is partially conserved in the antagonist bound form. In the (S)-ATPO complex, Ser⁷⁴¹ is connected to the nitrogen atom of the isoxazole ring through two water molecules, whereas a third water molecule seen in the (S)-glutamic acid complex is displaced by the tert-butyl group of (S)-ATPO. Although these two water molecules are conserved, their positions are slightly shifted due to the presence of the isoxazole ring (not shown).

Comparison of the binding mode of (S)-ATPO in iGluR5-S1S2 and in iGluR2-S1S2J (14) reveals a different location of the phosphonate group; see Fig. 5C, which results in slightly fewer direct contacts (Table 3). This may be caused by the fact that the iGluR5-S1S2:(S)-ATPO structure is more open than the iGluR2-S1S2J:(S)-ATPO structure and that a contact to a glycerol molecule is gained in the iGluR5 complex. Furthermore, in iGluR5-S1S2 the conformation of (S)-ATPO is stabilized via a water-mediated contact from the -carboxylate group to the phosphonate moiety. The 3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl moiety of (S)-ATPO functions as a spacer keeping domains 1 and 2 separated in iGluR5, thereby making it an antagonist as also previously observed in iGluR2 (14).

View larger version (48K): [in this window] [in a new window]   FIGURE 5. Binding mode of the antagonist (S)-ATPO in iGluR5-S1S2. A, Fo - Fc omit electron-density map of (S)-ATPO contoured at 3. Oxygen atoms are colored red, nitrogen atoms are blue, the phosphorus atom is orange, and carbon atoms are yellow. The chemical structure of (S)-ATPO is shown in Fig. 1. B, close-up view of the iGluR5-S1S2·(S)-ATPO complex including potential hydrogen bonds within 3.0 Å (dotted lines). Bonds of protein and ligand are shown in green and yellow, respectively. Water molecules are shown as red spheres. C, superposition (on domain 1 residues) of the structures of (S)-ATPO in complex with iGluR5-S1S2 and iGluR2-S1S2J (molA, PDB code 1N0T) (14). Bonds of protein and ligand are shown in green and yellow for iGluR5-S1S2 and in gray and black for iGluR2-S1S2J. Water molecules were omitted for clarity. D, superposition (on domain 1 residues) of the structures of iGluR5-S1S2 in complex with (S)-ATPO and UBP310 (molA, PDB code 2F34) (16). Bonds of protein and ligand are shown in green and yellow for iGluR5-S1S2:(S)-ATPO and in gray and black for iGluR5-S1S2: UBP310. The chemical structure of UBP310 is shown in Fig. 1.

(S)-ATPO displays selectivity toward AMPA receptors and iGluR5 and show no binding at iGluR6 (47). The most prominent difference between ligand-binding site residues of iGluR6 and iGluR1-4/iGluR5 is the presence of an alanine residue at position 689 in iGluR6, which is a serine residue at the corresponding position in the other receptors. This serine residue provides vital hydrogen bonds to the phosphonate group of (S)-ATPO via its side chain hydroxyl group, and such hydrogen bonds are abolished in iGluR6. Therefore, differences in the residue at position 689 (Ser/Ala) probably account for the marked selectivity profile.

The Dimerization Interface—iGluR5-S1S2 in complex with domoic acid forms a dimer similar to the well known dimer seen for iGluR2-S1S2J crystals (9, 48) and recently also observed in a mutant of iGluR5-S1S2 crystallized with the two UBP antagonists and (S)-glutamic acid (16). Overall, three areas within domain 1 (residues Ile⁵¹⁹-Thr⁵³⁵, Asp⁷⁶⁰-Lys⁷⁶², and Arg⁷⁷⁵-Met⁷⁹³) and one area within domain 2 (Thr⁶⁹²-Ile⁶⁹⁹) contribute to the dimer interface, resulting in a total interface accessible area of 1090 Ų made up of 30 residues (Fig. 6A). The single residues contributing mostly to the buried surface area are Tyr⁵²¹ (10%), Lys⁵³¹ (9%), Ile⁷⁸⁰ (7%), Gln⁷⁸⁶ (7%), and Glu⁷⁸⁷ (9%). In total nine residues are engaged in hydrogen bonding and/or salt bridge formation (Tyr⁵²¹ N-Glu⁷⁸⁷ OE-2, Glu⁵²⁴ OE-1-Lys⁵³¹ NZ, Phe⁵²⁹ O-Lys⁵³¹ NZ, Ser⁷⁶¹ N-Gln⁷⁸⁶ OE-1, and Arg⁷⁷⁵ NH-2-Asp⁷⁷⁶ OD-1).

It has previously been shown that domoic acid activates slowly desensitizing currents in iGluR5 channels, whereas (S)-glutamic acid activates rapidly desensitizing currents (49). To investigate interactions that serve to stabilize the dimer of the domoic acid complex relative to the (S)-glutamic acid complex (16), we performed a detailed analysis of the two structures (Fig. 6). As can be seen in Fig. 6A, the same residues are overall important for dimer formation but notably, the Arg⁷⁷⁵ NH-2-Asp⁷⁷⁶ OD-1 salt bridge and the Tyr⁵²¹ N-Glu⁷⁸⁷ OE-2 hydrogen bond are only present in the domoic acid complex (Fig. 6, B-D) due to conformational differences. Arg⁷⁷⁵ corresponds to the position of the R/G mRNA editing site previously demonstrated to affect desensitization of AMPA receptors (50, 51). Furthermore, mutation of Asp⁷⁷⁶ to a glycine residue in iGluR6 resulted in receptors with greatly accelerated desensitization kinetics relative to wild type receptors (52). Therefore, it can be hypothesized that the Arg⁷⁷⁵-Asp⁷⁷⁶ salt bridge in the domoic acid-bound iGluR5 complex is involved in stabilizing the dimer in the active form and thereby being partly responsible for the slower desensitization kinetics. The backbone hydrogen bond of Tyr⁵²¹ to the side chain of Glu⁷⁸⁷ is not present in the (S)-glutamic acid complex due to a small relocation of the individual monomers in this complex. Furthermore, Tyr⁵²¹ adopts different side chain conformations in the two structures (Fig. 6D). In AMPA receptors, the corresponding residue to Tyr⁵²¹ is a leucine (Leu⁴⁸³ in iGluR2), and mutation of this single amino acid into a tyrosine prevents receptor desensitization (53). Recently, it was shown that cysteine substitution of Tyr⁵²¹ and Leu⁷⁸³ in iGluR5 and corresponding residues in iGluR6 and iGluR7 creates kainate receptors locked in their active conformations by intermolecular disulfide cross-links (54). Thus, the position and conformation of Tyr⁵²¹ as well as the Tyr⁵²¹ N-Glu⁷⁸⁷ OE-2 hydrogen bond may be another factor contributing to the slower desensitization kinetics of domoic acid.

View larger version (53K): [in this window] [in a new window]   FIGURE 6. The dimerization interface. A, residues involved in dimerization of iGluR5-S1S2 in complex with (S)-glutamic acid (Glu), domoic acid (DA), and (S)-ATPO (ATPO) are shown and have been color coded according to their contribution to the interface; yellow, 0-1%; green, 1-6%; and red, 6-12%. Residues engaged in hydrogen bond formation (H), salt bridge formation (S), or both ($) are indicated below the sequences. B, the dimers of iGluR5-S1S2 shown in complex with domoic acid (green) and (S)-glutamic acid (gray) superimposed on domain 1 residues. Residues forming intermolecular bonds in the domoic acid complex but not in the (S)-glutamic acid complex are highlighted. The letter C indicates the Arg775-Asp776 interaction site of which a close-up view is shown in panel C, and the letter D indicates the Tyr521-Glu787 interaction site of which a close-up view is shown in panel D.

View larger version (28K): [in this window] [in a new window]   FIGURE 7. Histogram of domain openings (filled bars) and distances between equivalent TM1-TM2 linker residues of the dimer (open bars)in iGluR5-S1S2 (A) and in iGluR2-S1S2J (B) induced by different compounds. Domain openings were calculated relative to that of the (S)-glutamic acid complex. Distances between two Ile668 in the iGluR5 dimer (Ile633 in iGluR2) are shown. In A, GLU, the full agonist (S)-glutamic acid (PDB code 2F36) (16); DA, the partial agonist domoic acid; ATPO, the antagonist (S)-ATPO; UBP310, the antagonist UBP310 (PDB code 2F34) (16); and UBP302, the antagonist UBP302 (PDB code 2F35) (16). In B, GLU,(S)-glutamic acid (PDB code 1FTJ) (9); CPW, the partial agonist CPW-399 (PDB code 1SYH) (48); KA, the partial agonist kainic acid (PDB code 1FW0) (9); APO, the apo structure (PDB code 1FTO) (9); and ATPO, (S)-ATPO (PDB code 1N0T) (14). In all cases, molA was used.

An even larger domain opening of 28° is introduced upon binding of the antagonist (S)-ATPO in iGluR5-S1S2 (Fig. 7). This domain opening is only slightly smaller (1-2°) than the opening observed in the UBP302 and UBP310 structures with iGluR5 (16), which suggests that the antagonist structures of iGluR5 are good representatives for an apo iGluR5 structure. It appears that the span of domain opening is much larger in iGluR5 (30°) compared with what has been observed in iGluR2 (19°) (Fig. 7). For example, (S)-ATPO induces a 9° smaller domain opening in iGluR2-S1S2J compared with that seen in iGluR5-S1S2.

In Figs. 2 and 7A, the domain opening in iGluR5-S1S2 is correlated to the distance between the TM1-TM2 linker residues of the two protomers comprising the dimer. A similar correlation is also observed in iGluR2-S1S2J (Fig. 7B). However, much larger variation in the distances between TM1-TM2 linker residues are seen in iGluR5 compared with iGluR2. This suggests that iGluR5 undergoes a more drastic conformational change than iGluR2 upon activation by an agonist. Also, several D1-D2 interdomain contacts in iGluR5-S1S2 are gained upon binding of the full agonist (S)-glutamic acid as compared with the partial agonist domoic acid and especially to the antagonist (S)-ATPO (Table 4). In the structure of iGluR5-S1S2 in complex with (S)-glutamic acid, in total 12 interdomain hydrogen bonds/ionic interactions are seen of which three are conserved among all three structures (Glu⁷³⁸-Tyr⁷⁶⁴, Thr⁷⁴⁰-Tyr⁷⁶⁴, and Thr⁷⁴⁰-Trp⁷⁹⁹). The previously described interdomain "lock" between Glu⁴⁴¹-Ser⁷²¹ in iGluR5-S1S2 (10) and between Glu⁴⁰²-Thr⁶⁸⁶ in iGluR2-S1S2J (9) is not present in the complexes with domoic acid and (S)-ATPO. Instead, an interdomain contact is formed from the neighboring Glu⁴⁴²-Tyr⁷⁴⁴. Even though the number of iGluR5 complex structures is still sparse, the results suggest that a correlation exist between agonist efficacy at iGluR5 and TM1-TM2 linker distances (and domain closure) in the iGluR5-S1S2 construct as previously also observed for iGluR2 (48, 55).

	FOOTNOTES

 
The atomic coordinates and structure factors (codes 2PBW and 1VSO) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* The work was supported by grants from The Dansync Center for Synchrotron Radiation, The Drug Research Academy, The Ministry of Science, Technology, and Innovation, The Danish Medical Research Council, and The European Community-Access to Research Infrastructure Action of the Improving Human Potential Programme. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed. Tel.: 45-35-33-64-86; Fax: 45-35-33-60-40; E-mail: jsk{at}farma.ku.dk.

³ The abbreviations used are: iGluR, ionotropic glutamate receptor; AMPA, 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid; ATPO, 2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl]propionic acid; domoic acid, (2S,3S,4S)-3-carboxymethyl-4-[(1Z,3E,5R)-5-carboxy-1-methyl-hexa-1,3-dienyl]-pyrrolidine-2-carboxylic acid; iGluR2-S1S2J, ligand-binding core construct of iGluR2; iGluR5-S1S2, ligand-binding core construct of iGluR5; iGluR6-S1S2, ligand-binding core construct of iGluR6; TM, transmembrane; UBP302, (S)-1-(2-amino-2-carboxyethyl)-3-(2-carboxybenzyl)pyrimidine-2,4-dione; UBP310, (S)-1-(2-amino-2-carboxyethyl)-3-(2-carboxythiophene-3-yl-methyl)-5-methylpyrimidine-2,4-dione.

	ACKNOWLEDGMENTS

 
We kindly acknowledge B. Vestergaard for cryocooling of crystals.

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