Crystal structures of the kainate receptor GluR5 ligand binding core dimer with novel GluR5-selective antagonists

Abstract

Glutamate receptor (GluR) ion channels mediate fast synaptic transmission in the mammalian CNS. Numerous crystallographic studies, the majority on the GluR2-subtype AMPA receptor, have revealed the structural basis for binding of subtype-specific agonists. In contrast, because there are far fewer antagonist-bound structures, the mechanisms for antagonist binding are much less well understood, particularly for kainate receptors that exist as multiple subtypes with a distinct biology encoded by the GluR5-7, KA1, and KA2 genes. We describe here high-resolution crystal structures for the GluR5 ligand-binding core complex with UBP302 and UBP310, novel GluR5-selective antagonists. The crystal structures reveal the structural basis for the high selectivity for GluR5 observed in radiolabel displacement assays for the isolated ligand binding cores of the GluR2, GluR5, and GluR6 subunits and during inhibition of glutamate-activated currents in studies on full-length ion channels. The antagonists bind via a novel mechanism and do not form direct contacts with the E723 side chain as occurs in all previously solved AMPA and kainate receptor agonist and antagonist complexes. This results from a hyperextension of the ligand binding core compared with previously solved structures. As a result, in dimer assemblies, there is a 22 A extension of the ion channel linkers in the transition from antagonist- to glutamate-bound forms. This large conformational change is substantially different from that described for AMPA receptors, was not possible to predict from previous work, and suggests that glutamate receptors are capable of much larger movements than previously thought.

Figure 1.

Binding experiments with AMPA and kainate receptor S1S2 ligand-binding cores. A, Chemical structures of UBP310, UBP302, and the quinoxalinediones NBQX and CNQX. B, Summary of the results of radioligand displacement assays for the S1S2 constructs of GluR5, GluR6, and GluR2; K_d values are the mean ± SEM of three to six experiments per ligand. Selectivity was calculated from the ratio of the indicated K_d pairs. C, Displacement curves for binding of willardiine derivatives to GluR5 and GluR6. D, Displacement curves for binding of quinoxalinediones to GluR5 and GluR6; data points for C and D show the mean ± SEM.

Figure 2.

Selective antagonist action of UBP310. A–C, Inhibition of glutamate (Glu)-activated currents is shown for GluR2 (A), GluR5 (B), and GluR6 (C); UBP310 was applied at 3.25 μm for GluR5 and 32.5 μm for GluR2 and GluR6. D, Mean ± SD of responses from four to five cells per GluR subtype recorded as shown in A–C. E, Kinetics of recovery from inhibition by 3.25 μm UBP310 of the GluR5 response to glutamate.

Figure 3.

Ligand-induced domain closure for iGluR ligand binding cores. A, Stereoview of the GluR5 UBP310 (red), GluR2 ATPO (PDB code 1NOT; blue), and GluR5 glutamate (green) complexes superimposed using domain 1 Cα coordinates; the labels indicate the position of helices F, G, H, and I in domain 2. B, Analysis of domain closure defined as the rotation that is required to superimpose domain 2 for the indicated antagonist complexes following superimposition of domain 1 onto the GluR2, GluR5, or NR1 agonist complexes with glutamate or glycine as appropriate. C, Analysis of the rotation that is required to superimpose domain 2 for the indicated antagonist complexes following superimposition of domain 1 on the GluR5 UBP310 complex.

Figure 4.

Agonist-induced conformational changes in the GluR5 dimer assembly illustrated by superposition using domain 1 Cα coordinates of the UBP310 (red) and the glutamate (green) complexes. The top panel is viewed from the N terminus looking down the twofold axis; the bottom panel shows a view from the side after rotation by 90°. The relative movement of the linker regions, which connect domain 2 to the ion channel pore, is represented by black spheres connected by red and green dashed lines, which show the difference in position of the Cα coordinates of Ile653 in the two structures. This distance increases from 18.8 Å in the UBP310 complex to 40.9 Å in the glutamate complex.

Figure 5.

High-resolution crystal structure of the GluR5 UBP310 complex. A, Ribbon representation of the GluR5 S1S2 UBP310 complex colored by B-factor with a color ramp from blue to red over the B-value range 10–35; the ligand and side chains that interact with UBP310 are shown as ball-and-stick representations; the labels indicate the locations of α-helices; the Se peak energy anomalous difference electron density map contoured at 6 σ confirms the location of all nine methionine side chains, which are drawn as ball-and-stick representations. B, F_o-F_c omit electron density map calculated using data to 1.74 Å and contoured at 4.5 σ; atoms for UBP310 and the indicated waters were omitted from the F_c calculation. C, Stereoview of the ligand binding pocket in the UBP310 complex; the peptide chains from segments S1 and S2 are colored cyan and gold, respectively; UBP310 is shown as a transparent CPK model together with a ball-and-stick representation for the ligand and some side chains; water molecules are shown as green spheres; dashed lines indicate hydrogen bonds; alternative conformations for the side chains of Ser674, Met722 and Glu723 are shown as transparent objects.

Figure 6.

Subsite map of the GluR5 ligand-binding pocket. Schematic representations for the GluR5-binding pocket occupied by (S)-glutamate (A) and UBP310 (B). Hydrogen bond and ion pair sites generated by domains 1 and 2 are colored pink and blue, respectively; stripes indicate sites generated by both domains; sites of van der Waals contacts are indicated by hatched curved lines. In the glutamate complex, there are six trapped water molecules shown as numbered red spheres; four of these are maintained in the UBP310 complex. To make this figure, torsion angles in the UBP310 ligand were adjusted compared with the conformation found in the crystal structure to bring the heterocyclic rings into approximately the same plane for ease of illustration. A detailed explanation of the subsite maps is available as supplemental material (available at www.jneurosci.org).

Figure 7.

The Glu723 side chain undergoes a conformational switch. F_o-F_c omit electron density maps calculated using data to 1.87 and 2.11 Å for the GluR5 UBP302 (A) and the GluR5 glutamate (B) complexes contoured at 3.1 and 3.2 σ, respectively; atoms for Glu723, W7 and W8, and Asp745 through Tyr749 were omitted from the F_c calculation. Hydrogen bonds are shown by dashed lines. The UBP302 and glutamate ligands are drawn as yellow stick models. In the UBP302 structure, the Glu723 side chain was refined in two conformations: the first with 65% occupancy, makes a hydrogen bond with the side chain of Lys747, and the second, refined with 35% occupancy, is shown as a transparent stick figure and projects toward but does contact the ligand. In contrast, in the glutamate complex, the Glu723 side chain makes a hydrogen bond contact with the ligand α-amino group. Glu, Glutamate.