doi:10.1046/j.1432-1033.2002.03345.x
Eur. J. Biochem. 269, 6261–6270 (2002) (cid:2) FEBS 2002
Determinants of antagonist binding at the a-amino-3-hydroxy- 5-methyl-4-isoxazolepropionic acid receptor subunit, GluR-D Role of the conserved arginine 507 and glutamate 727 residues
Annukka Jouppila1, Olli T. Pentika¨ inen2, Luca Settimo2, Tommi Nyro¨ nen3, Jukka-Pekka Haapalahti1, Milla Lampinen1, David G. Mottershead1, Mark S. Johnson2 and Kari Keina¨ nen1 1Viikki Biocenter, Department of Biosciences (Division of Biochemistry) and Institute of Biotechnology, University of Helsinki, Finland, 2Department of Biochemistry and Pharmacy, A˚bo Akademi University, Turku, Finland, 3CSC – Scientific Computing Ltd, Espoo, Finland., Subdivision in Eur. J. Biochem. Neurochemistry
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 anta- gonist-specific, ranging from no interaction to weak elec- trostatic interactions involving indirect and direct hydrogen bonding with the antagonist molecule. These results under- line 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.
Keywords: AMPA; ionotropic glutamate receptor; molecu- lar modelling; Ro 48–8587; radioligand binding.
Previous structural and mutagenesis studies indicate that the invariant a-amino and a-carboxyl groups of glutamate receptor agonists are engaged in polar interactions with oppositely charged, conserved arginine and glutamate resi- dues in the ligand-binding domain of a-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 anta- gonists, 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,
Correspondence to K. Keina¨ nen, Department of Biosciences, PO Box 56, Viikinkaari 5D, 00014 University of Helsinki, Helsinki, Finland. Fax: + 358 919159068, Tel.: + 358 919159606, E-mail: kari.keinanen@helsinki.fi Abbreviations: AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazole- propionic acid; ATOA, (RS)-2-amino-3-[5-tert-butyl-3-(carboxy- methoxy)-4-isoxazolyl]propionic acid; ATPO, (RS)-2-amino-3- [5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl] propionic acid; CNQX, 6-cyano-7-nitro-quinoxaline-2,3-dione; DNQX, 6,7-dinitro- quinoxaline-2,3-dione; HEK, human embryonic kidney; PNQX, (1,4,7,8,9,10-hexahydro-9-methyl-6-nitropyrido[3,4-f]quinoxaline- 2,3-dione); NS 257, (1,2,3,6,7,8-hexahydro-3-(hydroxyimino)- N,N,7-trimethyl-2-oxobenzo[2,1-b:3,4-c¢] dipyrrole-5-sulfonamide); NS 1209, 8-methyl-5-(4-(N,N-dimethylsulfamoyl)phenyl)-6,7,8,9,- tetrahydro-1H-pyrrolo[3,2-h]-isoquinoline-2,3-dione-3-O-(4-hydroxy- butyric acid-2-yl)oxime; 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. (Received 12 August 2002, revised 15 October 2002, accepted 4 November 2002)
therapeutic potential as neuroprotective agents, detailed information regarding the structural basis of ligand recog- nition would be important for drug design and may also help in the understanding of the activation mechanism of the receptor [2]. a-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-selective glutamate receptors are multimeric ligand-gated channels, which mediate fast excitatory neurotransmission and, under pathological conditions, contribute to the excitotoxic actions of glutamate [1]. As it is believed that AMPA receptor antagonists may have
AMPA receptors are assembled from a set of four homologous subunit polypeptides, named GluR-A through -D, or alternatively, as GluR1–4, each with (cid:1) 900 amino acid residues and containing three predicted transmembrane segments and a pore loop as part of the channel structure [1,3]. Identification of homology between two segments (S1 and S2) in ionotropic glutamate receptor subunits and bacterial amino acid-binding proteins [4,5], and expression of a functional agonist binding site of the GluR-D and GluR-B subunits of the AMPA receptor as soluble S1–S2 fusion proteins, [6,7] paved the way to the recent structure determinations by Gouaux and coworkers, which provided the first atomic resolution view of a neurotransmitter binding site [8,9]. Crystal structures of S1–S2 constructs of the GluR-B (GluR2) AMPA receptor subunit with bound ligands show that the agonist ligands are buried deeply and are engaged in multiple polar interactions with the two lobes of the ligand-binding domain. The invariant a-aminocarb- oxyl moiety of the agonists is stabilized through ion pair and hydrogen bonding interactions involving the conserved residues Arg485 in Lobe 1 and Glu705 in Lobe 2 as predicted by site-directed mutagenesis [10]. (The numbering of GluR-D residues is according to the virtual translation
6262 A. Jouppila et al. (Eur. J. Biochem. 269)
(cid:2) FEBS 2002
Finland), and Fermentas (Vilnius, Lithuania). Anti-Flag M1 was obtained from Sigma. Anti-c-myc antibody was purified from the hybridoma cell line 9E10.2 originally obtained from American Type Culture Collection (ATCC CRL1729).
DNA constructs and recombinant baculovirus expression from the corresponding nucleotide sequences starting with the initiator methionine. This differs, by the length of the predicted signal peptide, from the numbering used by Gouaux and coworkers [8,9] for the mature GluR-B (GluR2) polypeptide [8,9].) The three hydrogen bonds of the a-amino group (to Pro478, Thr480, both in Lobe 1, and Glu705) show a nearly perfect tetrahedral organization around the central nitrogen. The distal acidic group of the agonists interacts exlusively with Lobe 2 [9].
Single point mutations were introduced into the flip isoform [17] of N-terminally Flag-tagged or C-terminally myc- tagged rat GluR-D in a pFASTBAC1 vector (BRL-Life Sciences) [6,18] by overlap extension PCR using mutagenic primers, followed by restriction fragment replacement. The presence of the mutations was verified by DNA sequencing. Recombinant baculovirus vectors were then prepared by using the Bac-to-Bac system according to the manufac- turer’s instuctions (Invitrogen Life Technologies, Carlsbad, CA, USA), and used to infect monolayers of Trichoplusia ni cells ((cid:2)High Five(cid:3), Invitrogen) grown in T25 or T175 tissue culture flasks at 27 (cid:3)C. SF900-II (Life Technologies, Paisley, UK), supplemented with penicillin (100 lgÆmL-1), strepto- mycin (lgÆmL-1) and amphotericin B (0.25 lgÆmL-1) was used as the culture medium. The cells were collected 4 days after infection by centrifugation (1000 g, for 10 min) and analysed by immunoblotting. The cell pellets were stored at )20 (cid:3)C until used for radioligand binding studies.
Importantly, the crystal structures of ligand-free GluR-B S1–S2 and one antagonist complex, 6,7-dinitroquinoxaline- 2,3-dione (DNQX), have also been obtained [9]. These complexes are slightly more open than the agonist com- plexes, suggesting that agonist-induced closure of the lobes may provide the driving force for channel opening [9]. Binding of the DNQX is mediated by polar contacts to both lobes and aromatic stacking with the side chain of a tyrosine residue. Consistent with pharmacophore models [11,12] predicting that the carbonyl oxygens and iminic nitrogens of the quioxalinedione antagonists, indispensable for activity, mimic the a-aminocarboxylate core of the agonists, the carbonyl oxygens of DNQX are hydrogen bonded to Arg485. Glu705 does not, however, participate directly in DNQX binding. Interestingly, however, this residue shows two different conformations, one of which is pointing directly below the quinoxalinedione ring and Glu705 is, in fact, the only binding site residue which shows major side chain reorientation between the agonist, antagonist and ligand-free complexes [9].
In some experiments, S1–S2 ligand-binding site con- structs were used instead of the full-length receptor. The Flag-tagged GluR-D S1–S2 constructs ((cid:2)wild-type(cid:3), E727D and E727A mutants), and their expression as soluble proteins secreted in the culture medium have been described previously [10].
Preparation of membranes
Considering the large structural variation of AMPA receptor antagonists [2,12], it can be expected that the receptor–antagonist interactions may show differences to those interactions seen in the DNQX complex structure. In the present study, we have analyzed the ligand-binding properties of mutated and wild-type homomeric GluR-D AMPA receptors in order to further characterize the roles of Arg507 and Glu727 (homologous to GluR-B residues Arg485 and Glu705, respectively) in antagonist binding. In order to separate the structural requirements necessary for antagonist binding from those important for agonist binding, we have used a high-affinity antagonist [3H]Ro 48-8587 [13] as a radioligand, in addition to [3H]AMPA.
M A T E R I A L S A N D M E T H O D S
Experimental materials
For radioligand binding experiments, membranes were prepared as follows. Insect cells were homogenized in five volumes of 20 mM Hepes, pH 7.4, 2.5 mM EDTA, 0.1 mM phenylmethanesulfonyl fluoride. The particulate fraction was pelleted (30 000 g, 25 min) and washed extensively by repeated homogenization and centrifugation in 20 mM Hepes, pH 7.4, 200 mM NaCl, 0.5 mM EDTA, 0.1 mM PMSF. Finally, the washed membranes were suspended in 20 mM Hepes, pH 7.4, 200 mM NaCl, 10% glycerol, 0.1 mM PMSF, and stored frozen or on ice until used in the assay. Some experiments were performed with receptor preparations that were solubilized in Triton X-100 as described previously [19] with results essentially identical to those obtained with membranes.
Radioligand binding assays
(PNQX)
CNQX, DNQX and kainic acid were obtained from RBI- Sigma (Natick, MA, USA) and L-glutamate was from Sigma (St Louis, MO, USA). [3H]AMPA (specific activity, 60 CiÆmmol)1) was from NEN Life Science Products [3H]Ro 48-8587 (specific activity, (Boston, MA, USA). 44 CiÆmmol)1) was obtained from Amersham Pharmacia Biotech (Buckinghamshire, UK). ATPO and ATOA [14] were obtained from P. Krogsgaard-Larsen, Royal Danish School of Pharmacy, Copenhagen, Denmark. NS 257 [15], NS 1209 [16], and 1,4,7,8,9,10-hexahydro-9-methyl-6-nitro- [11] were pyrido[3,4-f]quinoxaline-2,3-dione obtained from J. Drejer (NeuroSearch A/S, Glostrup, Denmark). Ro 48-8587 was obtained from R. Wyler and V. Mutel (F. Hoffmann-La Roche, Basel, Switzerland). Enzymes for molecular biology were purchased from New England Biolabs (Beverly, MA, USA), Finnzymes (Espoo, For the AMPA binding assays, the membranes were suspended in 30 mM Tris/HCl, pH 7.2, 100 mM potassium thiocyanate (KSCN), 2.5 mM CaCl2, 0.1% Triton X-100. For initial measurement of binding activity and for ligand competition experiments, 5 nM [3H]AMPA was used. For saturation analyses, [3H]AMPA was diluted to a specific activity of 12 CiÆmmol-1 with unlabelled R,S-AMPA and used at 1–300 nM concentrations. Membranes (20–250 lg protein) were incubated in a 0.5-mL volume for 1 h on ice with the radioligand, whereafter the reactions were
Role of E727 of GluR-D in antagonist binding (Eur. J. Biochem. 269) 6263
(cid:2) FEBS 2002
grid positions 0.25 A˚ apart terminated by adding 5 mL of ice-cold 30 mM Tris/HCl, pH 7.2, 100 mM KSCN, 2.5 mM CaCl2 followed by rapid filtration through Whatman GF/B filters with two 5-mL washes with ice-cold buffer. Nonspecific binding was determined in the presence of 1 mM L-glutamate. The filters were solubilized in OptiPhase 3 HiSafe (Wallac) and subjected to liquid scintillation counting.
ual interactions between atoms. Partial charges for the receptor model were calculated using the AMBER force field in SYBYL 6.6; and for ligands, the MMFF94 Force Field [28]. The interaction of a probe group (corresponding to each type of atom in the ligand) with a receptor model was calculated at in a (20 · 20 · 20) A˚ 3 box centered at the binding site using the program AUTOGRID in AUTODOCK 3.0. For each ligand, 10 separate docking simulations were performed.
[3H]Ro 48-8587 (0.5–2 nM final concentration) binding assays were performed in 50 mM Tris/HCl, pH 7.0 [13]. The filtration assay was performed as described for [3H]AMPA binding with the exception that 50 mM Tris/HCl, pH 7.0 was used as the buffer. Saturation binding analysis with [3H]Ro 48-8587 was performed by diluting the radioligand with increasing amounts of unlabelled compound.
Other assays. SDS/PAGE and Western blotting using anti-Flag M1 (Sigma Chemical, St. Louis, MO, USA) as the primary antibody, were performed as described previously [6,19]. Protein content of the samples was measured by using bicinchonic acid assay kit according to the manufac- turer’s instructions. (BCA, Pierce, Rockford, IL, USA). The ligand binding data were analyzed by nonlinear curve fitting using the PRISM 2.01 software (GraphPad Inc.). Student’s t-test was used for the statistical analyses.
R E S U L T S
Molecular modelling
Effects of mutations at residues Arg507 and Glu727 on binding of [3H]AMPA to GluR-D
Structural and mutagenesis studies on soluble ligand- binding domains indicate that Arg507 and Glu727 in the AMPA receptor subunit GluR-D, and the equivalent residues in the GluR-B subunit, serve as critical docking sites for the a-amino and a-carboxylate groups of agonist ligands. In the present study, we have examined the role of these residues in antagonist recognition in the full-length, membrane-bound AMPA receptor. Wild-type GluR-D and its R507K, E727D and E727Q mutants were expressed in recombinant baculovirus-infected Trichoplusia ni (High
Structural modelling. The three-dimensional structures of GluR-B S1–S2 ligand-binding core complexed with DNQX (PDB accession no. 1ftl [9]); was obtained from the Protein Data Bank [20]. The sequences of GluR-D and GluR-B were aligned by using MALIGN [21] in the BODIL Modeling Environment (Lehtonen J.V., Rantanen V.-V., Still, D.-J., Gyllenberg, M., and Johnson, M.S.; http://www.abo.fi/fak/ mnf/bkf/research/johnson/bodil.html, personal communi- cation) using a structure-based sequence comparision matrix [22]. The program MODELLER 4.0 [23] was used to construct a three-dimensional model structure by satisfying spatial restraints imposed on the GluR-D sequence by its alignment with the known GluR-B structure. At the same time, the structure of the ligand seen in the X-ray structure was built using MODELLER 4.0.
Ligand minimization. Ligands were built with the program SYBYL 6.6 (Tripos, St Louis, MO, USA) and energy minimized prior to docking to the receptor models using the TRIPOS Force Field and conjugate gradient method until the energy gradient was less than 0.05 kcalÆmol)1. Protona- tion of the polar groups was evaluated by comparison of the final three-dimensional structures with similar substructures obtained from the Cambridge Structural Data Bank (CSD System Documentation, 1992, Cambridge Crystallographic Data Centre, Cambridge, UK). To calculate the optimized structure for Ro 48–8587, standard Hartree–Fock (HF/3- 21G) methods in GAUSSIAN 98 [24] were used.
Fig. 1. Expression and [3H]AMPA binding activity of GluR-D mutant receptors. Wild-type and mutated epitope-tagged GluR-D AMPA receptors were expressed in recombinant baculovirus-infected High Five insect cells. (A) Immunoblot analysis showing the expression of Flag-tagged GluR-D (WT), GluR-D E727D, GluR-D E727Q and of myc-tagged GluR-D R507K. Noninfected cells were used as controls. All lanes were loaded with an equal amount ((cid:1) 10 lg) of membrane protein, and the blots were developed by using anti-Flag M1 or anti- myc 9E10 as primary antibodies as indicated. The positions of molecular size markers are shown at the right. (B) Binding of [3H]AMPA to mutant GluR-D receptors. Binding of 5 nM [3H]AMPA to insect cell membranes (50 lg protein for wild-type and E727 mutants; 100 lg for R507K) was determined in the presence (non- specific binding) and absence (total binding) of 1 mM glutamate. Specific binding was defined as the difference between total and non- specific binding. The values (mean ± SD) are from a representative assay performed in triplicate.
Receptor minimization. Polar hydrogens were added to the receptor using SYBYL 6.6. In order to optimize the intramolecular interactions in the receptor model, hydrogen atoms were minimized (keeping the rest of the model rigid) using amber charges [25,26].
Ligand docking. AUTODOCK 3.0 [27] is a semirigid docking program that considers the whole ligand molecule docking to the binding site and it is possible to choose the torsion angles of the ligand that are allowed to rotate (AutoTors), while the bond angles and bond lengths are kept fixed. The overall interaction energy between chemical species is estimated by considering both Lennard–Jones atom–atom potentials and electrostatic effects, summed for the individ-
6264 A. Jouppila et al. (Eur. J. Biochem. 269)
(cid:2) FEBS 2002
Fig. 3. Inhibition of [3H]AMPA binding by competitive antagonists. Wild-type (solid squares) and E727D mutant (open squares) GluR-D receptors were expressed in recombinant baculovirus-infected insect cells. Insect cell membranes were equilibrated with [3H]AMPA (5 nM) in the presence of increasing concentrations of unlabelled antagonist compounds, and the amount of bound [3H]AMPA was determined by rapid filtration. Displacement of [3H]AMPA binding is shown for DNQX, NS 1209, and Ro 48–8587. The curves represent a best fit to a one-site model obtained by nonlinear curve fitting.
Interaction of competitive antagonists with wild-type and E727D mutant receptors
Five) insect cells as homomeric, epitope-tagged receptors. In immunoblots, an (cid:1) 105-kDa immunoreactive band, corres- ponding to the size expected for a glycosylated GluR-D monomer, was observed in baculovirus-infected cells ex- pressing Flag-tagged GluR-D and the E727D and E727Q mutants, and the myc-tagged R507K mutant, but not in noninfected control cells (Fig. 1A). The presence of an additional and intense 90-kDa myc-immunoreactive band in cells expressing the R507K mutant suggests partial proteolysis or defective glycosylation with this mutant (Fig. 1A). To determine the ligand binding activity of the GluR-D mutants, membrane preparations from the bacu- lovirus-infected cells were subjected to a filtration binding assay by using [3H]AMPA (at 5 nM) as the radioligand. In accordance with a previous analysis conducted with the [3H]AMPA soluble ligand-binding domain of GluR-D, bound to wild-type GluR-D and to the E727D mutant but not to the R507K and E727Q mutants (Fig. 1B). In a saturation binding analysis, Kd values of 40 and 11 nM were obtained for the wild-type and E727D receptors, respect- ively. The corresponding Bmax values were 12.3 pmolÆmg)1 of protein for GluR-D and 14.7 pmolÆmg-1 for GluR-D E727D. In a competition binding assay, unlabelled L-glutamate exhibited a Ki value of 0.20 ± 0.06 lM for the wild-type GluR-D and 2.13 ± 0.13 lM for the GluR-D E727D mutant (mean ± SD; n ¼ 4). Kainate exhibited a Ki value of 2.10 ± 0.14 lM (mean ± SD; n ¼ 4) with the wild-type GluR-D, but displayed a drastically decreased affinity to the E727D mutant (> 1000-fold), consistent with earlier results obtained with the soluble ligand-binding domain [10]. A reliable value for the inhibitory affinity constant of kainate binding to E727D mutant receptor could not be obtained, but 3 mM kainate caused only 20–25% inhibition of [3H]AMPA binding (not shown).
Next, we determined the ability of competitive antagonists representing different structural types (Fig. 2) to inhibit [3H]AMPA binding to GluR-D and to the E727D mutant. All eight antagonist ligands inhibited [3H]AMPA binding to wild-type GluR-D in a concentration-dependent manner (Fig. 3, Table 1). With the exception of two compounds, Ro 48-8587 and NS 257, substituting an aspartate for the glutamate at position 727 of GluR-D, produced significant changes in the apparent binding affinitites of the antagonists (Table 1). The closely related quioxaline-2,3-diones CNQX and DNQX were ((cid:1) 20-fold) more potent inhibitors of [3H]AMPA binding at the mutant receptor, whereas an opposite effect was seen with PNQX, NS 1209 and the two AMPA-derivatives, ATPO and ATOA. In fact, the highest concentration of ATOA which was tested (1 mM), did not produce any clear inhibition of [3H]AMPA binding to the E727D mutant. The competition curves were monophasic and displayed Hill coefficients close to unity for all antagonists, except for ATOA and ATPO whose low affinity to the E727D mutant receptor prevented the determination of more complete displacement curves.
Fig. 2. Structures of AMPA receptor antagonists used in this study.
Binding of [3H]Ro 48-8587 to wild-type and mutated GluR-D receptors
As the E727Q and R507K mutant receptors were devoid of any [3H]AMPA binding activity, the effect of these muta- tions in antagonist recognition could not be studied by binding inhibition experiments. Therefore, we employed a recently introduced high-affinity antagonist radioligand, [3H]Ro 48-8587 [13], to analyze directly the antagonist binding properties of the mutant receptors. In a filtration assay using a single 1 nM concentration of [3H]Ro 48-8587, significant binding was observed to wild-type GluR-D, and to the E727D and E727Q mutants (Fig. 4A). In contrast, no binding above nonspecific background produced by mem- branes prepared from noninfected cells was seen with the R507K mutant (Fig. 4A). In saturation binding experi- ments, Kd values of 8.3 ± 0.2 and 7.0 ± 1.2 nM were obtained for the wild-type GluR-D and E727D, respect-
Role of E727 of GluR-D in antagonist binding (Eur. J. Biochem. 269) 6265
(cid:2) FEBS 2002
Table 1. Inhibition of [3H]AMPA binding to GluR-D and GluR-D E727D by competitive antagonists. Wild-type and E727D mutant GluR-D receptors were expressed in recombinant baculovirus-infected insect cells. Insect cell membranes were equilibrated with 5 nM [3H]AMPA in the presence of increasing concentrations of the indicated unlabelled antagonists. Non-specific binding, determined in the presence of 1 mM glutamate, was subtracted from all values. The specific Ki values were calculated by using the Cheng–Prusoff equation with Kd values of 40 and 11 nM for [3H]AMPA binding to the wild-type receptor and E727D, respectively. The values represent the mean ± SD from three to five independent determinations (n given in parenthesis). Statistical significance of the difference between the wild-type GluR-D and E727D mutant was determined by using Student’s t-test and is indicated as P-values. ND, not determined.
Antagonist
GluRD
GluRD E727D
P-value
CNQX DNQX PNQX NS 257 NS 1209 ATOA ATPO Ro 48–8587
0.433 ± 0.167 (3) 0.546 ± 0.180 (4) 0.334 ± 0.105 (5) 0.834 ± 0.110 (5) 0.087 ± 0.035 (4) 437 ± 118 (3) 67 ± 6 (3) 0.020 ± 0.004 (4)
0.022 ± 0.007 (4) 0.016 ± 0.006 (4) 2.57 ± 1.30 (4) 0.682 ± 0.092 (5) 1.61 ± 0.68 (4) > 1000a (3) > 1000b (3) 0.022 ± 0.003 (4)
0.0038 0.0011 0.0060 0.0464 0.0042 ND ND 0.454
a No inhibition was obtained at the highest concentration used: 97.7 ± 4.1% of the binding left at 1 mM ATOA. b Only a partial inhibition was obtained: 65.3 ± 3.5% of the binding left at 1 mM ATPO.
respectively (mean ± SD; ively, whereas a slightly higher Kd value, 20.0 ± 0.5 nM, was obtained for E727Q (mean ± SD; n ¼ 3; Fig. 4B).
n ¼ 3; 2.52 ± 0.15 lM, Fig. 5B), indicating that binding of these two antagonists (as with Ro 48–8587) can bind to the receptor in the presence of glutamine at position 727. Unfortunately, the small amount of labelled [3H]Ro 48-8587 available preven- ted more extensive displacement experiments.
Molecular model of GluR-D ligand-binding domain
analysis, Kd
In order to further substantiate the findings and to facilitate comparision with earlier work, we performed some [3H]Ro 48-8587 binding studies with the separately expressed S1–S2 ligand-binding domain of GluR-D (Fig. 4C). Consistent with the results obtained with the membrane-bound full-length receptors, [3H]Ro 48-8587 (at 2 nM concentration) bound specifically to the wild-type and E727D S1–S2 proteins, whereas the S1–S2 R507K mutant did not show binding activity (Fig. 4D). In a saturation binding 15.6 ± 6.6 and of values 13.0 ± 1.8 nM were obtained for the binding of [3H]Ro 48-8587 to wild-type S1–S2 and to the E727D mutant, respectively (mean ± SD; n ¼ 3). Furthermore, an S1–S2 protein carrying E727A mutation, previously shown to lack [3H]AMPA binding acitivity ([10,] Fig. 4D), did not show any [3H]Ro 48-8587 binding in the filtration assay (Fig. 4D). Ligand binding studies were complemented by docking simulations in order to help us understand the role of R507 and E727 in the interactions of the receptor with the different antagonists. First, a three-dimensional model of the ligand binding domain of GluR-D (S1– S2) was built using the 1.8 A˚ resolution structure of the GluR-B S1–S2-DNQX complex [9] as a template. The sequence identity between the molecules was high ((cid:1) 90%) with only one difference located near the binding site, Tyr703 in GluR-B being replaced by a phenylalanine in GluR-D.
Ligand-binding properties of E727Q mutant receptors Ligand docking
Quinoxalinediones. DNQX was docked automatically into the GluR-D receptor model resulting in very similar interactions to those present in the crystal structure of GluR-B S1–S2-DNQX complex, including formation of two hydrogen bonds with R507 (Fig. 6A). Glu727 does not form hydrogen bonds with DNQX and is likely to have the same two side chain orientations as the equivalent Glu705 has in the asymmetric unit of GluR- B S1–S2-DNQX complex. Both orientations could ac- commodate a weak electrostatic interaction with the DNQX ring structure, which carries a partial positive charge due to two strongly electron-withdrawing nitro groups. Furthermore, in the GluR-B S1–S2-DNQX complex [9], Tyr732 is seen to form a hydrogen bond with the 6-nitro group of DNQX or with Glu705, depending on the orientation of the side chain of Glu705. Similar interactions are likely to take place between DNQX and the corresponding residues, Glu727 and Tyr754, in GluR-D (Fig. 7A and B). Because earlier mutagenesis studies indicated that a negative charge at position 727 is necessary for agonist binding, the ability of the E727Q mutant receptor to bind [3H]Ro 48-8587 was of considerable interest, and therefore we examined further the [3H]Ro 48-8587 binding properties of homomeric E727Q receptors by using ligand competition experiments. Due to the limited amount of [3H]Ro 48-8587 available, only semiquantitative analyses were performed. Using a single 1 mM concentration of unlabelled ligand, the agonists, L-glutamate and kainate, were practically inactive as inhibitors of [3H]Ro 48-8587 binding to the E727Q receptor, whereas DNQX inhibited > 90% of the binding (Fig. 5A). As expected, all three ligands strongly inhibited [3H]Ro 48-8587 binding to wild-type GluR-D, whereas kainate was a significantly weaker inhibitor than glutamate and DNQX at E727D membranes, consistent with the dramatic decrease in kainate affinity caused by E727D mutation (Fig. 5A). Furthermore, DNQX and NS 1209 inhibited [3H]Ro 48-8587 binding to GluR-D E727Q membranes with Ki values of 0.033 ± 0.011 and
6266 A. Jouppila et al. (Eur. J. Biochem. 269)
(cid:2) FEBS 2002
Fig. 5. [3H]Ro 48-8587 binding properties of GluR-D E727Q mutant. [3H]Ro 48-8587 to membranes prepared from cells Binding of expressing wild-type and mutated GluR-D receptors was analyzed. (A) Inhibition of [3H]Ro 48-8587 (1 nM) binding to wild-type GluR-D and to E727D and E727Q mutant receptors by 1 mM glutamate, kainate and DNQX. The values are presented as percentage of the total binding obtained in the absence of competing ligands (equal to 100%) ± SD, and are from a representative experiment performed in triplicate. (B) Inhibition of [3H]Ro 48-8587 binding to GluR-D E727Q membranes by DNQX and NS 1209. The values represent means from a duplicate measurement. The displacement curves were obtained by nonlinear curve-fitting to a one-site model. The corresponding Ki values were 0.048 and 2.37 lM for DNQX (open circles) and NS 1209 (solid circles), respectively.
[3H]Ro 48-8587 to GluR-D mutant receptors. Fig. 4. Binding of Wild-type and mutated GluR-D AMPA receptors and soluble ligand- binding domains ((cid:2)S1–S2(cid:3)) were expressed in recombinant baculovirus- infected insect cells, and binding of [3H]Ro 48-8587 to membranes and to dialyzed culture supernanatants (in the case of S1–S2) was deter- mined. (A) total binding of 1 nM [3H]Ro 48-8587 to membrane sam- ples, each containing 50 lg of protein. The values represent mean ± SD. from a representative experiment performed in triplicate. indicated by binding of The level of nonspecific binding is [3H]Ro 48-8587 to wild-type GluR-D membranes in the presence of 1 mM glutamate ((cid:2)WT + Glu(cid:3)) and by total binding of [3H]Ro 48- 8587 to membranes prepared from uninfected cells ((cid:2)control(cid:3)). (B) Homologous displacement of [3H]Ro 48-8587 binding to wild-type GluR-D (solid squares), GluR-D E727D (open squares) and GluR-D E727Q (open circles) membranes by unlabelled Ro 48-8587. The values represent mean ± SD from a triplicate measurement. The displacement curves were obtained by nonlinear curve-fitting to a one- site model. Kd values of 9.7 ± 2.0, 8.3 ± 2.0 and 25.2 ± 2.0 nM were determined for the wild-type GluR-D, E727D and E727Q, respect- ively. (C) Schematic structure of the constructs for expression of full- length GluR-D and its ligand-binding domain (S1–S2), and an immunoblot showing the expression of wild-type and mutated epitope- tagged S1–S2 constructs as secreted 43-kDa anti-Flag-immunoreactive species in recombinant baculovirus-infected insect cells. The samples for immunoblotting correspond to 10 lL of culture supernatant. (D) Binding of [3H]Ro 48-8587 to wild-type and mutated S1–S2 proteins. Binding of [3H]Ro 48-8587 to extensively dialyzed culture superna- tants was determined by using filtration through polyethylene–imine- treated filters. The values correspond to total binding (mean ± SD) from a representative experiment conducted in triplicate. The level of nonspecific binding was determined for the wild-type S1–S2 in the presence of 1 mM glutamate (S1–S2-WT + Glu).
the pyrrolidine ring in NS 257 carry partial negative charges and interact with Arg507 in an analogous way to DNQX. No direct interaction is predicted between Glu727 and NS 257, consistent with the absence of any change in the ability of NS 257 to inhibit [3H]AMPA binding upon the mutation E727D (Table 1). In contrast, however, the hydroxyl group in the (cid:2)side chain(cid:3) of NS 1209, not present in NS 257, is ideally positioned to form a hydrogen bond with Glu727 in wild type GluR-D, but not with the E727D mutant, providing an explanation for the observed (cid:1) 18-fold decrease in affinity (Table 1). Interestingly, the carboxylate group of the 4-hydroxy-2-methyleneamino- oxybutyrate (cid:2)side chain(cid:3) of NS 1209 may be able to interact with the beginning of helix F, a partly positively charged region which binds the distal carboxylate groups of kainate and L-glutamate in the GluR-B crystal structures [8,9].
The docked conformation of CNQX closely resembles that of DNQX. Because of the bulky methylpiperidinic ring, the nitro group of PNQX cannot be positioned to form a hydrogen bond with Tyr754, and therefore a hydrogen bonding pattern similar to that illustrated in Fig. 7A, cannot form, and a situation illustrated in Fig. 7B is more likely. For the E727D mutant, the absence of a negatively charged group equivalent to the nitro group in DNQX and the presence of the positive charge would eliminate the possibility for interactions of the type shown in (Fig. 7C).
NS 257 and NS 1219. The docked conformation of the antagonist, NS 257, resembles that of PNQX (Fig. 6B). The carbonyl oxygen and the oxime (-C¼N-OH) substituents of ATPO and ATOA. Two antagonists, ATOA and ATPO, are structurally related to AMPA and carry a bulky t-butyl substituent at the 5-position of the isoxazole ring, and either a carboxymethoxy group (ATOA) or a phosphonomethoxy group (ATPO) at a position equivalent to the 3-hydroxyl group in AMPA. ATPO and ATOA dock to the receptor models in a fashion similar to that of AMPA in the GluR-B S1–S2-AMPA crystal structure (Fig. 6C). The essential
Role of E727 of GluR-D in antagonist binding (Eur. J. Biochem. 269) 6267
(cid:2) FEBS 2002
Fig. 7. Orientation of the side chain of Glu727 in (A) chain A and in (B) chain B of the GluR-B S1S2-DNQX complex [9] relative to the inter- acting nitro group of DNQX. The E727D mutation in the chain A structure is predicted to provide additional space for a water molecule, which mediates a new stabilizing interaction between the side chain carboxylate and Tyr754. Hydrogen–oxygen, nitrogen–oxygen and oxygen–oxygen distances are indicated.
Fig. 6. Stereo images of the docked conformations of antagonist ligands bound to GluR-D ligand-binding site generated by AUTODOCK. (A) DNQX, CNQX and PNQX; (B) NS 257 and NS 1209; (C) ATPO.
and enthalpy corrections to correspond to a temperature of 298.15 K and a pressure of 101 kPa) of the three forms indicate that a structure in which N1 is protonated (configuration III in Fig. 8A) instead of the carbonyl oxygen (I in Fig. 8A), is only slightly less favourable (+18 kJÆ- mol)1). In contrast, the third possible configuration in which N2 is protonated (II in Fig. 8A) is much less favoured (+77 kJÆmol)1). Configuration III can be docked to the binding site leading to hydrogen bonding between the carbonyl oxygen and Arg507, and the ring nitrogen and Arg507 (Fig. 8B). The other carbonyl oxygen may interact with Ser676 or with the N-terminus of the helix F, whereas the imidazole nitrogen of the ligand binds to the side chain hydroxyl of Thr708. The nitro group of Ro 48-8587 behaves
Fig. 8. Interaction of Ro 48-8587 with the receptor. (A) Optimized structures for three configurations of Ro 48-8587 (I, II, and III), cal- culated using standard Hartree–Fock (HF/3–21G) methods in Gaus- sian 98 [24], and the corresponding energy differences (relative to configuration I) are shown. (B) Stereo image of the docked confor- mation III of Ro 48-8587 bound to GluR-D ligand-binding site gen- erated by Autodock.
hydrogen bond and ion pair interactions with Arg507 and Glu727 are predicted to be closely similar to those in the AMPA complex. In contrast to AMPA, however, the receptor complexes of ATOA and ATPO would keep the receptor in a much more open form because the bulky t-butyl group and the acidic side chain substituents that contact the partly positively charged helix F region prevent the sterically further closure. In the E727D mutant, carboxylate group of the aspartate side chain cannot reach the a-amino-group of the ligand, thus explaining the remarkable decrease in the binding affinity (Table 1).
Ro 48–8587. Ro 48-8587 has the highest affinity of the tested antagonists, and its binding is little affected by the E727D or E727Q mutations (Fig. 4B, Table 1). Initial docking of Ro 48-8587 into the GluR-D model using an orientation in which its carbonyl groups would interact with Arg507, in a similar manner to quinoxaline dione antago- nists, resulted in an unfavourable situation in which a positively charged ring nitrogen of the ligand (N1 in Fig. 8A) would come close to Arg507 side chain. Quantum chemical calculations using Hartree–Fock (HF/3–21G) methods in Gaussian 98 [24] indicated, however, that the hydrogen atom (H in Fig. 8A) can actually be switched between two nitrogen atoms N1 and N2, and the carbonyl oxygen in the five-membered ring of the antagonist molecule (Fig. 8A). Electronic energies (including zero-point energy correction
6268 A. Jouppila et al. (Eur. J. Biochem. 269)
(cid:2) FEBS 2002
in the same way as the nitro group in the DNQX complex. There is no direct interaction with E727, consistent with the mutant binding data. Interestingly, we observed that for an alternative configuration (I in Fig. 8A) of Ro 48–8587, an opposite orientation in which the 8-nitro group would bind Arg507, may actually also provide some favourable hydro- gen bond interactions (not shown).
D I S C U S S I O N
[3H]Ro 48-8587 to homomeric GluR-D receptors was observed with the mutant, R507K. This finding, considered together with our ligand docking experiments, is consistent with essential hydrogen bond interactions between Arg507 and the carbonyl group of Ro 48–8587. Similar oxygen atoms, which are potentially isosteric with either one of the carbonyl oxygens of DNQX are present in all AMPA antagonists [12], suggesting that docking to Arg507 (or an equivalent arginine residue in other AMPA receptor subunits) may be a common feature for all (competitive) AMPA receptor antagonists. It must be noted, however, that the negative results in the present binding experiments may also be due to misfolding of the R507K mutant.
Role of Glu727 in agonist binding
In contrast to agonists, relatively little is known about the structural requirements for the interaction between the AMPA receptor and competitive antagonists. The recently determined crystal structures of the soluble ligand-binding core of the AMPA receptor subunit, GluR-B, included one complex with a bound antagonist, DNQX, and provided, for the first time, atomic resolution data on the antagonist– receptor interaction. The structures indicate a considerable overlap in the key binding residues between DNQX and agonist compounds, and suggest that antagonists like DNQX may not be able to stabilize a closed state of the ligand-binding domain [9].
All AMPA and kainate receptor subunits have a glutamate residue at a position corresponding to Glu727 in the GluR- D subunit, whereas NMDA receptor subunits carry an aspartate residue. Mutations introduced at this position in iGluR subunits have been reported to abolish channel activity [36,37] and ligand binding activity [10,32]. In the crystal structure of the agonist complexes of GluR-B S1–S2, the a-amino nitrogen of the agonist is at the centre of a tetrahedral arrangement of bonds which includes hydrogen bonds to Glu705, equivalent to Glu727 in GluR-D, and two residues from the opposite lobe of the ligand-binding domain, Pro478, and Thr480 and a covalent bond to the a-carbon of the agonist [8,9]. The arrangement is also consistent with an ion pair interaction between the a-amino group and the carboxylate side chain of Glu705, which is confirmed by the requirement for a negatively charged residue at position 727 for [3H]AMPA both in the S1–S2 ligand binding domain [10] and in the full-length GluR-D (present study).
The AMPA receptor radioligands most suitable for ligand binding studies, because of high binding affinity and like low nonspecific binding, are generally agonists [3H]AMPA [29] or 5-[3H]fluorowillardiine [30]. In muta- genesis studies, however, the exclusive use of agonist radioligands means that only those mutations that preserve the essential features required for agonist binding, can be analyzed. Availability of a high-affinity antagonist radio- ligand, like [3H]Ro 48–87587 [13] used in the present study, makes it possible to dissect the structural determinants needed for agonist binding from those required for inter- action with antagonist compounds [31]. Analysis of the effects of mutations, at two key agonist-binding residues in GluR-D, Arg507 and Glu727, on the binding of [3H]AMPA and [3H]Ro 48–87587 revealed significant differences in the roles these two residues play in the recognition of agonists the results indicate that vs. antagonists. Importantly, Arg507 plays an essential role in agonist and also antagonist binding, whereas Glu727 is crucial for the interaction of agonists with the receptor. Ligand diplacement assays were used to obtain more information on the relative effects of the mutations at Glu727 on the binding of antagonists representing widely different structural types. Below, we discuss the involvement of Arg507 and Glu727 in ligand interactions in the light of radioligand binding experiments and tentative antagonist docking models.
Role of Arg507
The interaction of the rigid ring imino nitrogen of kainate with the carboxylate side chain at position 727 is constrained by the partially closed state of the binding site already in the wild-type receptor, and therefore, the > 1000-fold loss in affinity in the E727D mutant is not unexpected. In contrast to kainate, the effects of the E727D mutation on the affinities for AMPA and glutamate were considerably smaller: glutamate bound to the mutant with a (10-fold) lower affinity, whereas the affinity for AMPA was slightly increased by the mutation. The structural basis for these opposing effects is not entirely clear, but inspection of the models provides a possible explanation, based on differences in the interactions of the water molecule bound to Asp727 and occupying the extra space created by the glutamate–aspartate mutation. In addition, this water is bound to the side chains of Thr502 and Ser676 in the binding site, and has capacity for one more hydrogen bond interaction. In the AMPA complex, an ideal hydro- gen bond can form with another water molecule (water #4 in [9]), which links the isoxazole hydroxyl with the base of helix F, whereas in the glutamate complex, the water faces the planar surface of the carboxylate group, and only a weak interaction would be possible.
study, no binding of either Arg507 in GluR-D is conserved across the superfamily of iGluR subunits and mutations at this position result in nonfunctional channels or a total loss or dramatic decrease of agonist binding [10,32–36]. In GluR-B S1–S2 crystal structures, the corresponding arginine residue (Arg485) forms hydrogens bonds with, and serves as a counterion for the a-carboxylate of the three different agonist molecules [8,9]. A similar essential role for this arginine is seen in the structure of the DNQX complex, where the carbonyl oxygen atoms of the antagonist, carrying partial negative charges, are both hydrogen bonded to Arg485. In the [3H]AMPA or present Role of Glu727 in antagonist binding In a competitive [3H]AMPA binding assay, the E727D mutation caused only modest ((cid:1) 3–25-fold) increases or
Role of E727 of GluR-D in antagonist binding (Eur. J. Biochem. 269) 6269
(cid:2) FEBS 2002
structural similarity with bacterial amino acid-binding proteins. Neuron 12, 1291–1300.
6. Kuusinen, A., Arvola, M. & Keina¨ nen, K. (1995) Molecular dis- section of the agonist binding site of an AMPA receptor. EMBO J. 14, 6327–6332.
7. Arvola, M. & Keina¨ nen, K. (1996) Characterization of the ligand- binding domains of glutamate receptor (GluR) -B and GluR-D subunits expressed in Escherichia coli as periplasmic proteins. J. Biol. Chem. 271, 15527–15532.
8. Armstrong, N., Sun, Y., Chen, G.Q. & Gouaux, E. (1998) Structure of a glutamate-receptor ligand-binding core in complex with kainate. Nature 395, 913–917.
9. Armstrong, N. & Gouaux, E. (2000) Mechanism for activation and antagonism of an AMPA-sensitive glutamate receptor: crystal structures of the GluR2 ligand binding core. Neuron 28, 165–181. 10. Lampinen, M., Pentika¨ inen, O., Johnson, M.S. & Keina¨ nen, K. (1998) AMPA receptors and bacterial periplasmic amino acid- binding proteins share the ionic mechanism of ligand recognition. EMBO J. 17, 4704–4711.
11. Bigge, C.F., Malone, T.C., Boxer, P.A., Nelson, C.B., Ortwine, D.F., Schelkun, R.M., Retz, D.M., Lescosky, L.J., Borosky, S.A., Vartanian, M.G., Schwarz, R.D., Campbell, G.W., Robichaud, L.J. & Wa¨ tjen, F. (1995) Synthesis of 1,4,7,8,9,10-hexahydro- 9-methyl-6-nitropyrido[3,4-f]-quinoxaline-2,3-dione and related quinoxalinediones: characterization of alpha-amino-3-hydroxy- 5-methyl-4-isoxazolepropionic acid (and N-methyl-D-aspartate) receptor and anticonvulsant activity. J. Med. Chem. 38, 3720–3740. 12. Nikam, S.S. & Kornberg, B.E. (2001) AMPA receptor antago-
nists. Curr. Med. Chem. 8, 155–170.
13. Mutel, V., Trube, G., Klingelschmidt, A., Messer, J., Bleuel, Z., Humbel, U., Clifford, M.M., Ellis, G.J. & Richards, J.G. (1998) Binding characteristics of a potent AMPA receptor antagonist [3H]Ro 48–8587 in rat brain. J. Neurochem. 71, 418–426.
14. Madsen, U., Bang-Andersen, B., Brehm, L., Christensen, I.T., Ebert, B., Kristoffersen, I.T., Lan, Y. & Krogsgaard-Larsen, P. (1996) Synthesis and pharmacology of highly selective carboxy and phosphono isoxazole amino acid AMPA receptor antago- nists. J. Med. Chem. 12, 1682–1691.
decreases in the binding affinities of most tested antagonists. A direct interaction with the antagonist takes place with two antagonists, ATOA and ATPO, which are closely related to AMPA, and therefore likely to bind in a similar manner. Ligand docking experiments indicated, however, that for the other antagonists tested, the effects (increased affinity for DNQX and CNQX; decreased affinity for PNQX and NS 1209) are indirect in nature. One factor contributing to the differences between these ligands is the charge and bulkiness of the ligand group that is positioned between Tyr472 and Tyr754: CNQX and DNQX have a negatively charged cyano or nitro group, Ro 48-8587 and NS 257 have a planar or almost planar and a (partially) positively charged group, and PNQX and NS 1209 have a positively charged tetragonal six-membered ring. In addition, occupation of the extra space resulting from the E727D mutation is achieved by two different mechanisms depending on the ligand (a) the presence of a water molecule and the hydrogen-bond pattern of the type shown in Fig. 7C; or (b) the e-amino group of Lys752 occupies this space forms hydrogen bonds to the side chain oxygens of Asp727, Thr502 and Tyr754. Two antag- onists, NS 257 and Ro 48-8587 did not discriminate between wild-type receptor and the E727D mutant receptor, indica- ting that they have no interaction with E727, consistent with docking experiments, or that the asparate can fully substitute for glutamate at that position.
In conclusion, our results suggest that Arg507 is engaged in strong hydrogen-bonding interactions with all antago- nists and that Glu727 plays only a minor role except for the AMPA derivatives, ATOA and ATPO. Analysis of mutations at position 727 in GluR-D highlight the importance of an ion-pair interaction with Glu727 (or equivalent residue) as an essential feature of agonist- receptor binding. Further studies, including electrophysio- logical analyses, are needed to clarify the role of Glu727 in receptor activation.
A C K N O W L E D G E M E N T S
15. Nielsen, E.O., Johansen, T.H., Watjen, F. & Drejer, J. (1995) Characterization of the binding of [3H]NS 257, a novel compet- itive AMPA receptor antagonist, to rat brain membranes and brain sections. J. Neurochem. 65, 1264–1273.
This work was supported by grants from the National Technology Agency (TEKES), the Academy of Finland, the Sigrid Juselius Foundation, and the Magnus Ehrnrooth Foundation. We thank Taru Kostiainen for technical assistance. We are grateful to Dr J. Drejer (NeuroSearch A/S, Glostrup, Denmark), Dr R. Wyler and Dr V. Mutel (F. Hoffmann-La Roche, Basel, Switzerland), and Professor P. Krogsgaard-Larsen (Royal Danish School of Pharmacy, Copenha- gen, Denmark) for their generosity in providing compounds used in this study.
R E F E R E N C E S
16. Nielsen, E.O., Varming, T., Mathiesen, C., Jensen, L.H., Moller, A., Gouliaev, A.H., Watjen, F. & Drejer, J. (1999) SPD 502: a water-soluble and in vivo long-lasting AMPA antagonist with neuroprotective activity. J. Pharmacol. Exp. Ther. 289, 1492–1501. 17. Sommer, B., Keina¨ nen, K., Verdoorn, T.A., Wisden, W., Burna- shev, N., Herb, A., Ko¨ hler, M., Takagi, T., Sakmann, B. & See- burg, P.H. (1990) Flip and flop: a cell-specific functional switch in glutamate-operated channels of the CNS. Science 249, 1580–1585. 18. Kuusinen, A., Abele, R., Madden, D.R. & Keina¨ nen, K. (1999) Oligomerization and ligand-binding properties of the ectodomain of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunit GluRD. J. Biol. Chem 274, 28937–28943.
1. Dingledine, R., Borges, K., Bowie, D. & Traynelis, S.F. (1999) The
glutamate receptor ion channels. Pharmacol. Rev. 51, 7–61.
19. Kuusinen, A., Arvola, M., Oker-Blom, C. & Keina¨ nen, K. (1995) Purification of recombinant GluR-D glutamate receptor produced in Sf21 insect cells. Eur. J. Biochem. 233, 720–726.
20. Berman, H.M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T.N., Weissig, H., Shindyalov, I.N. & Bourne, P.E. (2000) The Protein Data Bank. Nuceic. Acids Res. 28, 235–242.
2. Bra¨ uner-Osborne, H., Egebjerg, J., Nielsen, E.Ø., Madsen, U. & Krogsgaard-Larsen, P. (2000) Ligands for glutamate receptors: design and therapeutic prospects. J. Med. Chem. 43, 2609–2645. 3. Madden, D.R. (2002) The structure and function of glutamate
receptor ion channels. Nature Rev. Neurosci. 3, 91–101.
21. Johnson, M.S. & Overington, J.P. (1993) A structural basis for sequence comparisons. An evaluation of scoring methodologies. J. Mol. Biol. 233, 716–738.
4. Stern-Bach, Y., Bettler, B., Hartley, M., Sheppard, P.O., O’Hara, P.J. & Heinemann, S.F. (1994) Agonist selectivity of glutamate receptors is specified by two domains structurally related to bac- terial amino acid-binding proteins. Neuron 13, 1345–1357.
22. Johnson, M.S., May, A.C., Rodionov, M.A. & Overington, J.P. (1996) Discrimination of common protein folds: application of protein structure to sequence/structure comparisons. Methods Enzymol. 266, 575–598.
5. Kuryatov, A., Laube, B., Betz, H. & Kuhse, J. (1994) Mutational analysis of the glycine-binding site of the NMDA receptor:
6270 A. Jouppila et al. (Eur. J. Biochem. 269)
(cid:2) FEBS 2002
30. Hawkins, L.M., Beaver, K.M., Jane, D.E., Taylor, P.M., Sunter, D.C. & Roberts, P.J. (1995) Characterization of the pharmacology and regional distribution of (S) -[3H]-5-fluorowillardiine binding in rat brain. Br. J. Pharmacol. 116, 2033–2039.
31. Lampinen, M., Settimo, L., Pentika¨ inen, O.T., Jouppila, A., Mottershead, D.G., Johnson, M. & Keina¨ nen, K. (2002) Dis- crimination between agonists and antagonists by the a-amino- 3-hydroxy-5-methyl-4-isoxazole propionic acid –selective glutam- ate receptor. A mutation analysis of the ligand-binding domain of GluR-D subunit. J. Biol. Chem. 277, 41940–41947.
32. Paas, Y., Eisenstein, M., Medevielle, F., Teichberg, V.I. & Dev- illers-Thiery, A. (1996) Identification of the amino acid subsets accounting for the ligand binding specificity of a glutamate receptor. Neuron 17, 979–990.
23. Sˇ ali, A. & Blundell, T.L. (1993) Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779–815. 24. Frisch, M.J., Trucks, G.W., Schlegel, H.B., Scuseria, G.E., Robb, M.A., Cheeseman, J.R., Zakrzewski, V.G., Montgomery, J.A., Stratmann, R.E., Burant, J.C., Dapprich, S., Millam, J.M., Daniels, A.D., Kudin, K.N., Strain, M.C., Farkas, O., Tomasi, J., Barone, V., Cossi, M., Cammi, R., Mennucci, B., Pomelli, C., Adamo, C., Clifford, S., Ochterski, J., Petersson, G.A., Ayala, P.Y., Cui, Q., Morokuma, K., Malick, D.K., Rabuck, A.D., Raghav- achari, K., Foresman, J.B., Cioslowski, J., Ortiz, J.V., Stefanov, B.B., Liu, G., Liashenko, A., Piskorz, P., Komaromi, I., Gom- perts, R., Martin, R.L., Fox, D.J., Keith, T., Al-Laham, M.A., Peng, C.Y., Nanayakkara, A., Gonzalez, C., Challacombe, M., Gill, P.M.W., Johnson, B.G., Chen, W., Wong, M.W., Andres, J.L., Head-Gordon, M., Replogle, E.S. & Pople, J.A. (1998) Gaussian 98 (Revision A.7). Gaussian Inc., Pittsburgh, PA. 25. Weiner, S.J., Kollman, P.A., Nguyen, D.T. & Case, D.A. (1986) An all atom force field for simulations of proteins and nucleic acids. J. Comput. Chem. 7, 230–252.
33. Kawamoto, S., Uchino, S., Xin, K.Q., Hattori, S., Hamajima, K., Fukushima, J., Mishina, M. & Okuda, K. (1997) Arginine-481 mutation abolishes ligand-binding of the AMPA-selective gluta- mate receptor channel alpha1-subunit. Brain Res. Mol. Brain Res. 47, 339–344.
34. Laube, B., Hirai, H., Sturgess, M., Betz, H. & Kuhse, J. (1997) Molecular determinants of agonist discrimination by NMDA receptor subunits: analysis of the glutamate binding site on the NR2B subunit. Neuron 18, 493–503.
26. Cornell, W.D., Cieplak, P., Bayly, C.I., Gould, I.R., Merz,K.M. Jr, Ferguson, D.M., Spellmeyer, D.C., Fox, T., Caldwell, J.W. & Kollman, P.A. (1995) A second generation force field for the simulation of proteins, nucleic acids and organic molecules. J. Am. Chem. Soc. 117, 5179–5197.
27. Morris, G.M., Goodsell, D.S., Halliday, R.S., Huey, R., Hart, W.E., Belew, R.K. & Olson, A.J. (1998) Automated docking using a Lamarckian genetic algorithm and empirical binding free energy function. J. Comput. Chem. 19, 1639–1662.
35. Abele, R., Keina¨ nen, K. & Madden, D.R. (2000) Agonist-induced isomerization in a glutamate receptor ligand-binding domain. A kinetic and mutagenetic analysis. J. Biol. Chem. 28, 21355–21363. 36. Mano, I., Lamed, Y. & Teichberg, V.I. (1996) A venus flytrap mechanism for activation and desensitization of alpha-amino- 3-hydroxy-5-methyl-4-isoxazole propionic acid receptors. J. Biol. Chem. 271, 15299–15302.
28. Halgren, T.A. (1996) Merck molecular force field. II. MMFF94 van der Waals and electrostatic parameters for intermolecular interactions. J. Comput. Chem. 17, 520–552.
29. Honore´ , T., Lauridsen, J. & Krogsgaard-Larsen, P. (1982) The binding of [3H]AMPA, a structural analogue of glutamic acid, to rat brain membranes. J. Neurochem. 38, 173–178.
37. Williams, K., Chao, J., Kashiwagi, K., Masuko, T. & Igarashi, K. (1996) Activation of N-methyl-D-aspartate receptors by glycine: role of an aspartate residue in the M3–M4 loop of the NR1 sub- unit. Mol. Pharmacol. 50, 701–708.
