Structure of the kainate receptor subunit GluR6 agonist-binding domain complexed with domoic acid

Abstract

We report the crystal structure of the glycosylated ligand-binding (S1S2) domain of the kainate receptor subunit GluR6, in complex with the agonist domoate. The structure shows the expected overall homology with AMPA and NMDA receptor subunit structures but reveals an unexpected binding mode for the side chain of domoate, in which contact is made to the larger lobe only (lobe I). In common with the AMPA receptor subunit GluR2, the GluR6 S1S2 domain associates as a dimer, with many of the interdimer contacts being conserved. Subtle differences in these contacts provide a structural explanation for why GluR2 L483Y and GluR3 L507Y are nondesensitizing, but GluR6, which has a tyrosine at that site, is not. The structure incorporates native glycosylation, which has not previously been described for ionotropic glutamate receptors. The position of the sugars near the subunit interface rules out their direct involvement in subunit association but leaves open the possibility of indirect modulation. Finally, we observed several tetrameric assemblies that satisfy topological constraints with respect to connection to the receptor pore, and which are therefore candidates for the native quaternary structure.

Fig. 1.

GluR6 topology and sequence alignment. (A) The topology of iGluR subunits makes it possible to express the GluR6 agonist-binding domain comprising S1 and S2 as a soluble polypeptide, removing both the N-terminal domain and membrane domains M1–M4. Ligand (white) is shown bound to the S1S2 domain. SP, signal peptide. (B) The regions of the GluR6 S1S2 polypeptide modeled in the structure are shown, aligned to equivalent residues from the GluR2 and NR1 structures. The numbering for GluR6 and NR1 is for the protein including signal sequence; the numbering for GluR2 is for the mature polypeptide, to conform with common usage in the literature. Secondary structure elements in GluR6 are indicated by rectangles (α-helices) and arrows (β-strands). Gaps, introduced on the basis of structural proximity, are indicated by dashes, and dots denote residues not visible in the structures. Primary sequence homology is indicated by medium and light gray for identical and conserved residues, respectively. Linker sequences are shown in black. Positions of the four potential N-linked glycosylation sites are shown by boxed numbers above the sequence. Residues interacting with the domoate molecule via ionic interactions through the side chain (asterisks) or main chain (pluses) and via hydrophobic interactions (dashes) are indicated above the sequence (see also Fig. 3).

Fig. 2.

The structure determined for GluR6 S1S2 (protomer a) is shown in two views ≈90° apart. S1 is colored yellow, and S2 is blue. The N-acetylglucosamine–fucose sugar moieties modeled at N423 (gray) and domoate (orange) are shown in stick representation. Labels indicate the relative location of the chains within the full-length subunit, lobes I and II, and the construct N and C termini. *, helix 774–788.

Fig. 3.

Views of the domoate-binding pocket; domoate carbon atoms are colored orange and GluR6 carbons, green. (A) Electron density for domoate and the main interacting residues in protomer a is shown with a stick representation of the final model structure. The fourfold NCS averaged density (contouredat1σ) was calculated with sigmaa-weighted coefficients and phases from the initial molecular replacement solution (four promoters and no ligands). Only the main-chain carboxyl of P516 is shown for clarity. (B) The observed ligand–receptor interactions in protomer a are shown in schematic form (generated by using ligplot; see ref. 37). Dotted gray lines indicate polar interactions and red hatching hydrophobic contacts. Main-chain atoms are labeled, as are substituted pyrrolidine ring carbons. (C) Electron density and model structure are shown for residues E686 to T692 (main chain only for T692) and domoate. (D) The same view is shown in stereo for an alignment of the GluR6 (green carbons), GluR2-AMPA (purple carbons), and GluR2-KA (gray carbons) structures (for clarity, the side chains of residue GluR6 E686 and its GluR2 equivalent are omitted).

Fig. 4.

Views of the AMPA-like dimer interface in GluR6 and GluR2. The interface between dimers is highlighted by shading. Structures were aligned over lobe I only. (A) Density is shown around residues K531 and T779 (protomer c on the left) and residues E524 F529 (protomer e on the right). A polar interaction is observed between the T779 side chain and the K531 main chain nitrogen. (B) The same view of GluR6 (green residue labels) is shown aligned with GluR2 protomers a and c (AMPA complex, carbons, and residue labels in gray). GluR2 N747 forms hydrogen bonds with both the K493 main chain and the E486 side chain. (C) An interaction observed only in GluR6 is shown for the same protomer pairs (GluR6, green cartoon; GluR2, purple cartoon), looking down the twofold axis from lobe I. GluR6 residues R775 and D776 (yellow; green labels) and GluR2 residues G743 and G743 and N744 (orange; purple label) are highlighted. There is a clear movement of helix 774–788 (arrowheads) compared with the equivalent helix in GluR2 (742–755). (D) The environment around GluR6 residue Y521 in the dimer formed by protomer a with itself (green carbons) is shown compared with the equivalent residues in wild-type GluR2 (gray carbons) and GluR2 L483Y (1LB8; red carbons). Residues Y521 to K525 are shown on the left (residues L/Y483 to E487 in GluR2) and I780 to Q784 (L748 to K752 in GluR2) on the right. For clarity, side chains are shown only for the first and last residues in each chain.