Abstract
The crystal structures of the ligand-binding core of the agonist complexes of the glutamate receptor-B (GluR-B) subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-selective glutamate receptor indicate that the distal anionic group of agonist molecules are stabilized by interactions with an N-terminal region of an alpha-helix (helix F) in the lobe 2 ("domain 2," Armstrong, N., and Gouaux, E. (2000) Neuron 28, 165-181) of the two-lobed ligand-binding domain. We used site-directed mutagenesis to further analyze the role of this region in the recognition of both agonists and antagonists by the AMPA receptor. Wild-type and mutated versions of the ligand-binding domain of GluR-D were expressed in insect cells as secreted soluble polypeptides and subjected to binding assays using [(3)H]AMPA, an agonist, and [(3)H]Ro 48-8587 (9-imidazol-1-yl-8-nitro-2,3,5,6-tetrahydro[1,2,4]triazolo[1,5-c] quinazoline-2,5-dione), a high affinity AMPA receptor antagonist, as radioligands. Single alanine substitutions at residues Leu-672 and Thr-677 severely affected the affinities for all agonists, as seen in ligand competition assays, whereas similar mutations at residues Asp-673, Ser-674, Gly-675, Ser-676, and Lys-678 selectively affected the binding affinities of one or two of the agonists. In striking contrast, the binding affinities of [(3)H]Ro 48-8587 and of another competitive antagonist, 6,7-dinitroquinoxaline-2,3-dione, were not affected by any of these alanine mutations, suggesting the absence of critical side-chain interactions. Together with ligand docking experiments, our results indicate a selective engagement of the side chains of the helix F region in agonist binding, and suggest that conformational changes involving this region may play a critical role in receptor activation.
Highlights
The crystal structures of the ligand-binding core of the agonist complexes of the glutamate receptor-B (GluR-B) subunit of the ␣-amino-3-hydroxy-5-methyl-4isoxazole 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 ␣-helix in the lobe 2 (“domain 2,” Armstrong, N., and Gouaux, E. (2000) Neuron 28, 165–181) of the two-lobed ligand-binding domain
The N-terminal end of an ␣-helix 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
We introduced alanine substitutions into the ligand-binding domain (S1S2) of the AMPA receptor subunit GluR-D in an area that corresponds to a loop (Leu-672–Ser-674) and the N-terminal end of helix F (Gly-675–Lys-678) in the ligandbinding core of GluR-B [4, 5]
Summary
The crystal structures of the ligand-binding core of the agonist complexes of the glutamate receptor-B (GluR-B) subunit of the ␣-amino-3-hydroxy-5-methyl-4isoxazole 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 ␣-helix (helix F) in the lobe 2 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 [4, 5]. 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 [9], 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 an-
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