Structure of the S1S2 glutamate binding domain of GLuR3

Abstract

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

Figure 1

Structure of GluR3_i S1S2 domain bound to glutamate with a number of residues discussed in the text shown. The S1 sequence is shown in cyan and the S2 sequence in blue. The four residues that differ between flip and flop forms are colored red.

Figure 2

Binding sites for (A) AMPA bound to GluR2_o (1ftm; cyan) and GluR3_i (yellow) and (B) glutamate bound to GluR2_o (cyan) and GluR3_i (yellow). Water residues in the GluR2_o structures are shown in orange and those in the GluR3_i structures are shown in red. Electron density maps for GluR3_i (C) and GluR2_o (D) bound to glutamate. The alternative conformations of M708 are illustrated for GluR2_o. (E) The regions of GluR2_o (cyan) and GluR3_i (yellow) undergoing a rotation of the peptide backbone are illustrated. The S652/G451 hydrogen bond is longer in GluR3_i.

Figure 3

(A) The dimeric structure of GluR3 bound to glutamate. The helices are labeled as in Armstrong et al.. Residues participating in contacts across the dimer interface are labeled. P632 (yellow) flanks the artificial linker region of the construct and is the point that connects to the linkers to the ion channel. The four residues that differ in flip and flop are shown in shades of red. (B) The dimer interface at the S754 (flip)/N754 (flop) interaction with S729. Both can form hydrogen bonds, but the structure of the water surrounding the hydrogen bond differs.

Figure 4

(A) Binding of [³H]AMPA to the S1S2 domains of GluR2_o and GluR3_i. The K_D of binding differed by less than 2-fold: 19 ± 2 nM for GluR2 and 43 ± 5 nM for GluR3. Armstrong and Gouaux reported a K_D of 24.8 ± 1.8 nM for [³H]AMPA binding to GluR2. (B) Structures of the ligands used in the binding studies, (C) The inhibition of [³H]AMPA binding by agonists, partial agonists, and antagonist to the S1S2 domains of GluR2_o and GluR3_i. Except for willardiine, the IC₅₀ values were within 2-fold for the two subtypes: (ligand, GluR2 IC₅₀/GluR3 IC₅₀; IC₅₀ expressed in μM) fluorowillardiine, 0.0040±.0009/0.0062±0.0014; iodowillardiine, 0.46±0.05/0.79±0.14; Cl-HIBO, 5.0±0.3/55±4; willardiine, 3.1±0.2/0.99±0.18; UBP277, 135±12/69±10. In all cases, GluR2_o is shown with filled symbols, and GluR3_i is shown with open symbols.