Binding site and ligand flexibility revealed by high resolution crystal structures of GluK1 competitive antagonists

Abstract

The availability of crystal structures for the ligand binding domains of ionotropic glutamate receptors, combined with their key role in synaptic function in the normal and diseased brain, offers a unique selection of targets for pharmaceutical research compared to other drug targets for which the atomic structure of the ligand binding site is not known. Currently only a few antagonist structures have been solved, and these reveal ligand specific conformational changes that hinder rational drug design. Here we report high resolution crystal structures for three kainate receptor GluK1 antagonist complexes which reveal new and unexpected modes of binding, highlighting the continued need for experimentally determined receptor-ligand complexes.

Figure 1

Structures and binding assays for GluK1 competitive antagonists. (A) UBP315 and UBP318 are willardiine derivatives with bromine substitutions on the thiophene and uracil rings respectively; LY466195 is a decahydroisoquinoline with fluorine substitutions on the pyrrolidine ring. (B) Radioligand displacement curves for the purified GluK1 ligand binding domain showing competition between ³[H]-L-glutamate and three antagonists, fit with single binding site isotherms of 33 ± 4 nM, 186 ± 23 nM and 38 ± 7 nM for UBP315, UBP318 and LY466195, respectively; values are mean ± SEM of triplicate measurements.

Figure 2

GluK1 antagonists produce different extents of domain closure. (A) Cα traces for crystal structures of GluK1 complexes with UBP315 (yellow), UBP318 (green) and LY466195 (red), superimposed using domain 1 Cα coordinates; with the exception of loop 1 and loop 2 the structures are essentially identical, but show different extents of domain closure, illustrated by movements of helices F, G and H in domain 2. (B) Extent of domain opening relative to glutamate (GluK1 and GluA2) and glycine (GluN1) bound crystal structures for a series of competitive antagonists illustrates that the ligand binding domain adopts a range of conformations for all three iGluR families.

Figure 3

Electron density maps for crystal structures of the GluK1 UBP315 and UBP318 complexes. (A) Stereoview at 1.8 Å resolution shows an Fo-Fc omit map contoured at 3.5 sigma (dark green) for which UBP315 atoms were omitted from the Fc calculation, and 2mFo-DFc maps for side chains (pale green) and water molecules (blue). (B) Stereoview of electron density maps at 1.8 Å resolution for the UBP318 complex prepared as described above for UBP315,

Figure 4

Crystal structures and subsite maps for the GluK1 UBP 315 and UBP318 complexes. (A) Stereoview of the GluK1 UBP315 complex; the ribbon diagram showing secondary structure elements for domains 1 and 2 is colored cyan and gold, respectively; waters are shown as green spheres; hydrogen-bonds and ion pair interactions are represented as dashed lines. The side chain of Glu426 forms a halogen bond with Br25 of the ligand. (B) Stereoview of the GluK1 UBP318 complex colored as above; note the different conformation of the Glu723 and S726 side chains, rearrangement of solvent networks, and the change in orientation of the thiophene ring compared to the UBP315 complex. (C) Subsite map showing schematic representations of UBP315 with the GluK1 binding pocket; 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 red lines. To make this figure, torsion angles in the UBP315 ligand were adjusted compared to the conformation found in the crystal structure to bring the heterocyclic rings into approximately the same plane for ease of illustration. (D) Subsite map showing schematic representations of UBP318 with the GluK1 binding pocket.

Figure 5

Crystal structure of the GluK1 LY466195 complex. (A) Fo-Fc map at 1.58 Å resolution contoured at 3.5 sigma; atoms for LY466195 from subunit D were omitted from the Fc calculation. (B) Stereoview of the GluK1 LY466195 complex for subunit D colored as for Fig. 3; the 4,4-difluoro-pyrrolidine adopts a down conformation, and both halogen atoms project towards helix H in the back of ligand binding pocket. (C) Stereoview of the GluK1 LY466195 complex for subunit A in which the ligand 4,4-difluoro-pyrrolidine group adopts an up conformation and projects out of the ligand binding pocket. (D) Subsite map showing schematic representations of LY466195 with the GluK1 binding pocket of subunit D colored and oriented to match the representation shown in Figs. 3 and 4.