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• W2023647928 abstract "The crystal structures of the ligand-binding core of the agonist complexes of the glutamate receptor-B (GluR-B) subunit of the α-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 α-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 [3H]AMPA, an agonist, and [3H]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 [3H]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. The crystal structures of the ligand-binding core of the agonist complexes of the glutamate receptor-B (GluR-B) subunit of the α-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 α-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 [3H]AMPA, an agonist, and [3H]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 [3H]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. glutamate receptor α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid 6,7-dinitroquinoxaline-2,3-dione 9-imidazol-1-yl-8-nitro-2,3,5,6- tetrahydro[1,2,4]triazolo[1,5-c] quinazoline-2,5-dione The ligand-binding site of ionotropic glutamate receptors is composed of two extracellular segments, S1 and S2, homologous to bacterial amino acid-binding proteins (1Stern-Bach Y. Bettler B. Hartley M. Sheppard P.O. O'Hara P.J. Heinemann S.F. Neuron. 1994; 13: 1345-1357Abstract Full Text PDF PubMed Scopus (395) Google Scholar, 2Kuryatov A. Laube B. Betz H. Kuhse J. Neuron. 1994; 12: 1291-3000Abstract Full Text PDF PubMed Scopus (334) Google Scholar, 3Kuusinen A. Arvola M. Keinänen K. EMBO J. 1995; 14: 6327-6332Crossref PubMed Scopus (173) Google Scholar). Recent determination of the crystal structure of an S1S2 fusion protein of the GluR-B1 (GluR2) α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunit as complexed with ligands (4Armstrong N. Sun Y. Chen G.Q. Gouaux E. Nature. 1998; 395: 913-917Crossref PubMed Scopus (603) Google Scholar, 5Armstrong N. Gouaux E. Neuron. 2000; 28: 165-181Abstract Full Text Full Text PDF PubMed Scopus (792) Google Scholar) provided the first atomic resolution view into a neurotransmitter-binding pocket and confirmed the earlier predictions of a close structural and functional similarity to the bacterial proteins (1Stern-Bach Y. Bettler B. Hartley M. Sheppard P.O. O'Hara P.J. Heinemann S.F. Neuron. 1994; 13: 1345-1357Abstract Full Text PDF PubMed Scopus (395) Google Scholar, 2Kuryatov A. Laube B. Betz H. Kuhse J. Neuron. 1994; 12: 1291-3000Abstract Full Text PDF PubMed Scopus (334) Google Scholar, 3Kuusinen A. Arvola M. Keinänen K. EMBO J. 1995; 14: 6327-6332Crossref PubMed Scopus (173) Google Scholar, 6Paas Y. Eisenstein M. Medevielle F. Teichberg V.I. Devillers-Thiery A. Neuron. 1996; 17: 979-990Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 7Wo Z.G. Oswald R.E. Mol. Pharmacol. 1996; 50: 770-780PubMed Google Scholar, 8Laube B. Hirai H. Sturgess M. Betz H. Kuhse J. Neuron. 1997; 18: 493-503Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar, 9Lampinen M. Pentikäinen O. Johnson M.S. Keinänen K. EMBO J. 1998; 17: 4704-4711Crossref PubMed Scopus (60) Google Scholar). The agonists kainate, glutamate, and AMPA are engaged in multiple polar and van der Waals contacts that stabilize a closed state of the two-lobed binding domain (4Armstrong N. Sun Y. Chen G.Q. Gouaux E. Nature. 1998; 395: 913-917Crossref PubMed Scopus (603) Google Scholar, 5Armstrong N. Gouaux E. Neuron. 2000; 28: 165-181Abstract Full Text Full Text PDF PubMed Scopus (792) Google Scholar). The charged α-aminocarboxylate group of glutamate and AMPA and the pyrrolidine carboxyl and imino groups of kainate are accommodated by hydrogen bonds and ion pair interactions with the oppositely charged side chains of Arg-485 and Glu-705, respectively. The distal negatively charged group of the agonists, the carboxylmethyl group of kainate, the γ-carboxylate of glutamate, or the isoxazole ring hydroxyl of AMPA, make close polar contacts with the base of an α-helix (“helix F”) in the lobe 2, but the exact hydrogen bonding patterns differ between agonists (4Armstrong N. Sun Y. Chen G.Q. Gouaux E. Nature. 1998; 395: 913-917Crossref PubMed Scopus (603) Google Scholar, 5Armstrong N. Gouaux E. Neuron. 2000; 28: 165-181Abstract Full Text Full Text PDF PubMed Scopus (792) Google Scholar). In contrast to the agonist complexes, the ligand-free apo form and one antagonist complex (6,7-dinitroquinoxaline-2,3-dione (DNQX)) of GluR-B S1S2 assume a more open state, suggesting that agonist activity is based on their ability to induce a slight closure of the lobes (5Armstrong N. Gouaux E. Neuron. 2000; 28: 165-181Abstract Full Text Full Text PDF PubMed Scopus (792) Google Scholar). Although the stereochemical features of the agonist-receptor interaction revealed by the crystal structure are largely supported by mutagenesis data, not much is yet known on how the receptor discriminates between agonists and antagonists or on how structurally different antagonists interact with the receptor. In particular, it is currently unclear to what extent the contacts antagonists make with the receptor overlap those made by agonists. Although previous mutagenesis work has identified amino acid residues that affect antagonist affinities in the AMPA receptor, for example Lys-471 and Tyr-472 in GluR-D (9Lampinen M. Pentikäinen O. Johnson M.S. Keinänen K. EMBO J. 1998; 17: 4704-4711Crossref PubMed Scopus (60) Google Scholar), no mutations that would selectively affect only agonists or antagonists have been reported. As a step toward understanding the structural basis of discrimination between agonists and antagonists by AMPA receptors, we have analyzed the ligand binding properties of the mutated GluR-D ligand-binding domain constructs by using [3H]AMPA, an agonist, and [3H]Ro 48-8587, a high affinity AMPA receptor antagonist (10Mutel V. Trube G. Klingelschmidt A. Messer J. Bleuel Z. Humbel U. Clifford M.M. Ellis G.J. Richards J.G. J. Neurochem. 1998; 71: 418-426Crossref PubMed Scopus (23) Google Scholar), as radioligands. The results from our ligand binding and molecular docking experiments indicate a selective engagement of the helix F region in agonist binding, suggesting that conformational transitions involving this region are involved in receptor activation. [3H]AMPA (specific activity, 60 Ci/mmol) was obtained from PerkinElmer Life Sciences. [3H]Ro 48-8587 (specific activity, 44 Ci/mmol) was obtained from Amersham Biosciences. Unlabeled RS-AMPA and kainate were obtained from Sigma. Unlabeled Ro 48-8587 is a kind gift from Drs René Wyler and Vincent Mutel (Hoffmann-La Roche). The residues from Leu-672 to Lys-678 in the ligand-binding domain of GluR-D (flip isoform; see Ref.11Sommer B. Keinänen K. Verdoorn T.A. Wisden W. Burnashev N. Herb A. Köhler M. Takagi T. Sakmann B. Seeburg P.H. Science. 1990; 249: 1580-1585Crossref PubMed Scopus (976) Google Scholar) were individually replaced by alanine residues by using a PCR-assisted mutagenesis approach. The plasmid pK503-4 (3Kuusinen A. Arvola M. Keinänen K. EMBO J. 1995; 14: 6327-6332Crossref PubMed Scopus (173) Google Scholar) encoding the wild-type GluR-D ligand-binding domain (S1S2) carrying an N-terminal FLAG epitope and a C-terminal Myc tag was used as a template. The mutations were generated by using a common 5′-primer (5′-GGGTTGCTAGCTATAAATATGAGGATTATTTGCAGG-3′) and the following mutation-specific 3′-primers: 5′-GGTGTTGAATTCTTTTGTTGACCCAGAATCGGCTGTTCCATAGGCAATTTCTG-3′ (L672A), 5′-GGTGTTGAATTCTTTTGTTGACCCAGAGGCAAGTGTTCCATAGGCAATTT-3′ (D673A), 5′-GGTGTTGAATTCTTTTGTTGACCCGGCATCAAGTGTTCCATAGGCAA-3′ (S674A), 5′-GGTGTTGAATTCTTTTGTTGAGGCAGAATCAAGTGTTCCATAGG-3′ (G675A), 5′-GGTGTTGAATTCTTTTGTGGCCCCAGAATCAAGTGTTCCAT-3′ (S676A), 5′-GGTGTTGAATTCTTTGGCTGACCCAGAATCAAGTGTTC-3′ (T677A), and 5′-GGTGTTGAATTCGGCTGTTGACCCAGAATCAAGTG-3′ (K678A). The PCR products were digested with NcoI and EcoRI and ligated into a similarly treated pK503-4 vector. The presence of all of the designed mutations was verified by DNA sequencing. Recombinant baculoviruses were generated by using the Bac-to-Bac system (Invitrogen) and used to infect Trichoplusia ni (High Five, Invitrogen) cells growing in T-25 tissue culture flasks as described previously (9Lampinen M. Pentikäinen O. Johnson M.S. Keinänen K. EMBO J. 1998; 17: 4704-4711Crossref PubMed Scopus (60) Google Scholar). The cells and culture media were harvested 3–4 days after infection. The culture supernatants were extensively dialyzed against 20 mm Tris-HCl, pH 7.2, 2.5. mm CaCl2, 100 mm KSCN (AMPA binding buffer; see Ref. 12Honoré T. Drejer J. J. Neurochem. 1988; 51: 457-461Crossref PubMed Scopus (116) Google Scholar), or 50 mm Tris-HCl, pH 7.0 (Ro 48-8587 binding buffer; see Ref. 10Mutel V. Trube G. Klingelschmidt A. Messer J. Bleuel Z. Humbel U. Clifford M.M. Ellis G.J. Richards J.G. J. Neurochem. 1998; 71: 418-426Crossref PubMed Scopus (23) Google Scholar) to remove endogenous glutamate. Dialyzed samples (20–100 μl) were equilibrated with [3H]AMPA (5 nm) or [3H]Ro 48-8587 (1 nm) in the respective binding buffers for 1 h on ice in a total volume of 0.5 ml. The bound and free radioactivity were separated by rapid filtration through polyethyleneimine-treated GF/B (Whatman) glass fiber filters. The 3H radioactivity on the filters was measured by liquid scintillation counting in OptiPhase (Wallac). Apparent affinities for AMPA, Ro 48-8587, kainate, glutamate, and DNQX were determined by using competition binding experiments. The radioligand binding data were analyzed by using the GraphPad Prism nonlinear curve-fitting software. For statistical analysis, an unpaired Student's t test was used. The four three-dimensional structures of GluR-B S1S2 complexed with AMPA (Protein Data Bank accession code: 1FTM), (2S-(2α, 3β,4β))-2-carboxy-4-(1-methylethenyl)-3-pyrrolidineacetic acid (kainate; 1ftk) l-glutamate (1FTJ), and DNQX (1FTL) were obtained from the Protein Data Bank (5Armstrong N. Gouaux E. Neuron. 2000; 28: 165-181Abstract Full Text Full Text PDF PubMed Scopus (792) Google Scholar, 13Berman H.M. Westbrook J. Feng Z. Gilliland G. Bhat T.N. Weissig H. Shindyalov I.N. Bourne P.E. Nucleic Acids Res. 2000; 28: 235-242Crossref PubMed Scopus (26931) Google Scholar). The sequence alignment of human GluR-D and rat GluR-B was made using MALIGN (14Johnson M.S. Overington J.P. J. Mol. Biol. 1993; 233: 716-738Crossref PubMed Scopus (266) Google Scholar, 15Johnson M.S. May A.C. Rodionov M.A. Overington J.P. Methods Enzymol. 1996; 266: 575-598Crossref PubMed Google Scholar) in the Bodil Molecular Modeling Environment (www.abo.fi/fak/mnf/bkf/research/johnson/bodil.html) 2J. Lehtonen, V.-V. Rantanen, D.-J. Still, M. Gyllenberg, and M. S. Johnson, unpublished results. using a structure-based sequence comparison matrix (14Johnson M.S. Overington J.P. J. Mol. Biol. 1993; 233: 716-738Crossref PubMed Scopus (266) Google Scholar, 15Johnson M.S. May A.C. Rodionov M.A. Overington J.P. Methods Enzymol. 1996; 266: 575-598Crossref PubMed Google Scholar). The program HOMODGE in Bodil was used to construct a three-dimensional model structure by keeping the side-chain conformations of all identical residues fixed and by maintaining the corresponding torsion angles of similar aligned residues. Ligands were built with the program Sybyl 6.6 (Tripos, St. Louis, MO). Each ligand structure was 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 kcal/mol. Protonation of polar groups was checked by comparison of the final three-dimensional structures with similar substructures obtained from the Cambridge Structural Data Bank (29Allen F.H. Kennard O. Chem. Des. Automation News. 1993; 8: 31-37Google Scholar). Polar hydrogens were added to the receptor models using SYBYL. To optimize the intramolecular interactions in the receptor models, hydrogen atoms were minimized (keeping the rest of the model rigid) using AMBER charges. AUTODOCK 3.0 (16Morris G.M. Goodsell D.S. Halliday R.S. Huey R. Hart W.E. Belew R.K. Olson A.J. J. Comput. Chem. 1998; 19: 1639-1662Crossref Scopus (9037) Google Scholar) is a semirigid docking program that considers the whole ligand molecule in the conformational search at the binding site, and it is possible to choose the torsion angles of the ligand that are allowed to rotate, whereas bond angles and bond length 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 individual interactions between atoms. Partial charges for the receptor model were calculated using the AMBER force field (17Cornell W.D. Cieplak P. Bayly C.I. Gould I.R. Merz Jr., K.M. Ferguson D.M. Spellmeyer D.C. Fox T Caldwell J.W. Kollman P.A. J. Am. Chem. Soc. 1995; 117: 5179-5197Crossref Scopus (11491) Google Scholar, 18Weiner S.J. Kollman P.A. Nguyen D.T. Case D.A. J. Comput. Chem. 1986; 7: 230-252Crossref PubMed Scopus (3589) Google Scholar) in SYBYL. For ligands, the semiempirical molecular orbital method PM3 (19Stewart J.J.P. J. Comput. Aided Mol. Des. 1990; 4: 1-105Crossref PubMed Scopus (2792) Google Scholar) from the MOPAC package within SYBYL was used. This semiempirical method considers only the valence-shell electrons explicitly. The interaction of a probe group (corresponding to each type of atom in the ligand) with a receptor model was calculated, using the program AUTOGRID in the AUTODOCK package, at grid position 0.25 Å apart in a 20 × 20 × 20 Å3 box centered at the binding site. For each ligand, 10 separate docking simulations were performed. Atomic and chemical group affinity maps in the binding site were calculated with GRID 19 (20). This program evaluates interaction energies between various test probes and the target protein molecule, predicting where a particular chemical probe interacts most favorably within the receptor-binding site. For the calculation, a cubic box (15 × 15 × 15) Å3was centered in the binding site, and residue side-chain flexibility was allowed. SDS-PAGE and Western blotting were performed as described previously (3Kuusinen A. Arvola M. Keinänen K. EMBO J. 1995; 14: 6327-6332Crossref PubMed Scopus (173) Google Scholar, 9Lampinen M. Pentikäinen O. Johnson M.S. Keinänen K. EMBO J. 1998; 17: 4704-4711Crossref PubMed Scopus (60) Google Scholar). Protein concentrations were measured by using the bicinchoninic acid assay (Pierce) as described by the manufacturer. The N-terminal end of an α-helix (helix F) and the preceding residues in the S2 segment of the ligand-binding domain of the AMPA receptor form a major region of contact for the distal anionic group of agonists (subsites D, E, and F in the crystal structure of GluR-B S1S2-agonist complexes; see Ref. 5Armstrong N. Gouaux E. Neuron. 2000; 28: 165-181Abstract Full Text Full Text PDF PubMed Scopus (792) Google Scholar). In the present study, we have used site-directed mutagenesis and direct binding experiments with radiolabeled agonist and antagonist compounds to further examine the role of this region in ligand recognition. Mutated GluR-D S1S2 constructs having an alanine substituting individually for residues from Leu-672 to Lys-678 were expressed in recombinant baculovirus-infected High Five cells and appeared as a 40–42-kDa anti-FLAG immunoreactive species in the culture supernatants (Fig.1). Comparison of secreted and cell-associated immunoreactivities revealed no major differences in the expression levels or in the relative amount of secreted protein, suggesting that the mutations do not cause any gross misfolding of the respective S1S2 proteins (Fig. 1 B). The relative amounts of the 42-kDa- and 40-kDa- secreted species, probably representing two differently glycosylated forms, were variable but did not show any consistent differences between the mutant and the wild-type polypeptide. To separately address the agonist and antagonist binding properties of the mutated S1S2 proteins, we used [3H]AMPA, a high affinity agonist, and [3H]Ro 48-8587, a recently described high affinity competitive AMPA receptor antagonist (10Mutel V. Trube G. Klingelschmidt A. Messer J. Bleuel Z. Humbel U. Clifford M.M. Ellis G.J. Richards J.G. J. Neurochem. 1998; 71: 418-426Crossref PubMed Scopus (23) Google Scholar), as radioligands. First, the ability of the S1S2 mutants to bind [3H]AMPA was determined in a qualitative binding assay using a single 100 nm concentration, which is saturating for the wild-type S1S2. Clear binding activity was observed for the mutants D673A, S674A, S676A, and K678A, whereas no binding above the nonspecific background measured in the presence of 1 mmglutamate was observed for the mutants L672A, G675A, and T677A (Fig.2). The [3H]AMPA binding properties of the four active mutants were analyzed in more detail by using competition experiments with unlabeled AMPA andl-glutamate. The apparent affinities for AMPA (D673A, S676A, K678A) or glutamate (S674A, K678A) were found to be slightly decreased (Table I).Table I[3H]AMPA binding properties of GluR-D S1S2 mutantsConstructK dAMPAK il-GlunmμmS1S224.8 ± 3.65 (n = 3)0.279 ± 0.365 (n = 3)L672ANB–D673A65.1 ± 18.8* (n = 4)1.870 ± 0.369** (n = 3)S674A32.6 ± 3.78* (n = 4)NDG675ANB–S676A92.6 ± 24.1** (n = 4)0.596 ± 0.181* (n = 4)T677ANB–K678A103 ± 20.8** (n = 3)0.840 ± 0.124** (n = 3)Apparent binding affinities are indicated as K d andK i values obtained from radioligand displacement experiments (see “Experimental Procedures”). Each value represents a mean ± S.E. from three to four experiments as indicated. NB, no measurable binding; ND, not determined; –, not determinable because of the absence of [3H]AMPA binding. Statistical significance of the difference to wild-type S1S2 is indicated as p values: *, p < 0.05; **, p< 0.01 (Student's unpaired t test). Open table in a new tab Apparent binding affinities are indicated as K d andK i values obtained from radioligand displacement experiments (see “Experimental Procedures”). Each value represents a mean ± S.E. from three to four experiments as indicated. NB, no measurable binding; ND, not determined; –, not determinable because of the absence of [3H]AMPA binding. Statistical significance of the difference to wild-type S1S2 is indicated as p values: *, p < 0.05; **, p< 0.01 (Student's unpaired t test). Next, we employed [3H]-labeled Ro 48-8587 to directly determine the ligand binding properties of the mutants irrespective of their [3H]AMPA binding activity. In a filtration binding assay, [3H]Ro 48-8587 bound to GluR-D S1S2 with high affinity (K i 15.6 nm; Fig.3, Table II), in agreement with nanomolar binding affinity reported for rat brain membranes (10Mutel V. Trube G. Klingelschmidt A. Messer J. Bleuel Z. Humbel U. Clifford M.M. Ellis G.J. Richards J.G. J. Neurochem. 1998; 71: 418-426Crossref PubMed Scopus (23) Google Scholar). Expectedly, the binding was displaced by unlabeled glutamate (K i 1.25 μm), kainate (K i 3.72 μm), and DNQX (K i 0.20 μm) (Fig. 3, Table II). In contrast to [3H]AMPA binding, all seven alanine substitution mutants bound [3H]Ro 48-8587 with similar high affinity (K i values for Ro 48-8587 ranging from 9.5 to 26 nm but did not differ from the wild-type value in a statistically significant manner (Table II). The relatively intact antagonist binding was not unique to Ro 48-8587, as another antagonist of a different structural type, DNQX, also inhibited [3H]Ro 48-8587 binding to the mutated S1S2 constructs displaying closely similar K i values for the wild-type and mutants (ranging from 0.11 to 0.32 μm; Table II). Glutamate inhibited [3H]Ro 48-8587 binding to the wild-type and most of the mutated S1S2 proteins with aK i value in the low micromolar range except for two of the non-AMPA-binding mutants, L672A (K i for glutamate 97 μm; 78-fold decrease in affinity) and T677A (K i 4.8 mm; 3840-fold decrease). Interestingly, the G675A mutant, which did not bind [3H]AMPA, displayed a similar K i value for glutamate as the wild-type S1S2 (Table II). Kainate inhibited [3H]Ro 48-8587 binding to all S1S2 proteins at micromolar concentrations. Significantly reduced affinities were observed with the L672A (26-fold) and T677A (13-fold) mutants, whereas somewhat smaller affinity decreases were seen with the G675A and K678A mutations (TableII).Table II[3H]Ro 48–8587 binding properties of GluR-D S1S2 mutantsConstructK d Ro 48–8587K i AMPAK il-GluK ikainateK i DNQXnmμmμmμmμmS1S215.6 ± 6.560.027 ± 0.0051.25 ± 0.6273.72 ± 1.070.200 ± 0.091L672A20.1 ± 5.8947.4 ± 6.4297.3 ± 14.0***95.2 ± 29.9**0.136 ± 0.037D673A9.53 ± 2.31ND2.55 ± 1.491.63 ± 0.20*0.282 ± 0.146S674A23.8 ± 3.45ND1.02 ± 0.072.36 ± 0.3380.218 ± 0.033G675A18.4 ± 6.925.98 ± 2.981.33 ± 0.40422.5 ± 8.21**0.214 ± 0.163S676A26.1 ± 9.98ND3.49 ± 1.07***4.49 ± 1.570.323aThe value is from a single experiment.T677A13.8 ± 1.45230aThe value is from a single experiment.4800 ± 1400***49.2 ± 6.22***0.112 ± 0.029K678A14.6 ± 0.473ND2.14 ± 0.4419.05 ± 2.64**0.169 ± 0.017Apparent binding affinities are indicated as K d orK i values obtained from radioligand displacement experiments (see “Experimental Procedures”). Each value represents a mean ± S.E. from three to five experiments unless otherwise indicated. ND, not determined. Statistical significance of the difference to wild-type S1S2 is indicated as p values: *, p < 0.05; **, p< 0.01; ***, p < 0.001 (Student's unpairedt test).a The value is from a single experiment. Open table in a new tab Apparent binding affinities are indicated as K d orK i values obtained from radioligand displacement experiments (see “Experimental Procedures”). Each value represents a mean ± S.E. from three to five experiments unless otherwise indicated. ND, not determined. Statistical significance of the difference to wild-type S1S2 is indicated as p values: *, p < 0.05; **, p< 0.01; ***, p < 0.001 (Student's unpairedt test). In additional experiments, the ability of unlabeled AMPA to displace [3H]Ro 48-8587 binding to the “AMPA-negative” S1S2 mutants was determined. L672A, G675A, and T677A mutant S1S2 polypeptides were tested. In all three cases, severe, ∼2000-fold (L672A), ∼300-fold (G675A), and ∼11,000-fold decreases in the binding affinity were observed (Table II), consistent with the failure to see any [3H]AMPA binding activity in the filtration assay. Thus, mutations in the helix F region seem to selectively affect agonist affinities in an agonist-specific manner, with little or no effect on the binding of two different competitive antagonists. This striking behavior is illustrated in Fig. 3, which shows the inhibition of [3H]Ro 48-8587 binding to L672A, G675A, and T677A by unlabeled agonist and antagonist compounds. These findings suggest that in contrast to AMPA receptor agonists, neither DNQX nor Ro 48-8587 does interact with the side chains of residues 672–678. Molecular modeling was used to interpret the ligand binding data in terms of the three-dimensional structure of the receptor. Four models of GluR-D S1S2, differing in the degree of domain closure, were built on the basis of the GluR-B S1S2 structures with bound l-glutamate, AMPA, kainate, and DNQX (5Armstrong N. Gouaux E. Neuron. 2000; 28: 165-181Abstract Full Text Full Text PDF PubMed Scopus (792) Google Scholar). The sequence identity between GluR-D and GluR-B over the S1S2 region is 89.6% with only one difference near the binding site: a phenylalanine residue (Phe-724) in GluR-D replaces a tyrosine residue in GluR-B. AMPA,l-glutamate, and kainate were automatically docked to the respective GluR-D S1S2 model structures (Fig.4, A–C). The modeled conformations of these ligands are close to those derived from the corresponding crystal structures: the root mean squared deviations over all ligand atoms between the GluR-D model and the structures of GluR-B S1S2 complexes with AMPA, l-glutamate, and kainate were 0.51, 0.64, and 0.60, respectively. Alanine substitutions at three positions, residues Leu-672, Gly-675, and Thr-677, resulted in clear changes in agonist binding, whereas similar mutations of Asp-673, Ser-674, Ser-676, and Lys-678 had only minor effects (Tables I and II). Generally, we found these findings to be in good agreement with the model. Three (Asp-673, Ser-674, and Lys-678) of the four residues that show no gross alterations when mutated do not have any contacts with the agonists. The side chains of Asp-673, Ser-674, and Lys-678 point away from the binding cavity, but a salt bridge with a local stabilizing effect may form between Asp-673 and Lys-678. Such an indirect role is in agreement with the observed, relatively minor affinity changes when these residues were mutated. The main chain amide nitrogens of Ser-676 and Thr-677 carry a partial positive charge due to their location at the N terminus of the helix F dipole and are hydrogen-bonded to the distal anionic group of the agonists. The side chain of Ser-676, however, does not have a direct contact with any of the agonists. Instead, its hydroxyl group is linked via a water bridge (Fig. 4 D, w2) to Glu-727 in GluR-D, perhaps explaining the modest decreases in the binding affinities of agonists caused by the S676A mutation (Tables Iand II). Replacement of Leu-672 by alanine resulted in a total loss of [3H]AMPA binding and dramatically lowered the affinities of glutamate and kainate when measured in the [3H]Ro 48-8587 competition binding assay (Table II). In the GluR-B S1S2 structures with bound agonist, the corresponding residue, Leu-650, is found in two different conformations: one present in the AMPA and glutamate complexes and the other one observed in the kainate complex. We suggest that the latter conformation is likely to be relevant for Leu-672 in GluR-D in all three agonist complexes. This conformation of Leu-672 will ideally pack against the aromatic ring of Phe-727 (which substitutes for Tyr-702 in GluR-B), resulting in slightly better contact with the hydrophobic parts of the agonist ligands (Figs. 4,A–C, and 5). In the case of AMPA, the total loss of binding with the L672A mutation is in agreement with extensive hydrophobic contacts between leucine and both the methylene group and the aromatic/hydrophobic part of the isoxazol ring of AMPA (Fig. 4 A). Furthermore, the considerable extra space created by the alanine substitution is not easily accommodated by side-chain rearrangements and would provide a less than ideal location for a water molecule. Similarly, the impaired binding affinity ofl-glutamate for the L672A mutant is consistent with a total or partial loss of hydrophobic contacts with the two methylene groups of the agonist. Interestingly, the bulky isopropenyl group of kainate is predicted to interact favorably with an alanine side chain at position 672, in agreement with the observed moderate (26-fold) decrease in binding affinity with L672A (Table II). The effects of the alanine substitution at residue Gly-675 were remarkably specific for AMPA binding: despite the total loss of [3H]AM" @default.
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