Crystal structure and association behaviour of the GluR2 amino-terminal domain

Abstract

Fast excitatory neurotransmission is mediated largely by ionotropic glutamate receptors (iGluRs), tetrameric, ligand-gated ion channel proteins comprised of three subfamilies, AMPA, kainate and NMDA receptors, with each subfamily sharing a common, modular-domain architecture. For all receptor subfamilies, active channels are exclusively formed by assemblages of subunits within the same subfamily, a molecular process principally encoded by the amino-terminal domain (ATD). However, the molecular basis by which the ATD guides subfamily-specific receptor assembly is not known. Here we show that AMPA receptor GluR1- and GluR2-ATDs form tightly associated dimers and, by the analysis of crystal structures of the GluR2-ATD, propose mechanisms by which the ATD guides subfamily-specific receptor assembly.

Figure 1

The isolated GluR1 and GluR2 ATDs are dimers. (A) Sedimentation equilibrium studies of the GluR1 ATD. Lower panel: absorbance data (red dots) fit to a monomer–dimer model (black line), showing calculated populations of monomer (green) and dimer (blue). The loading concentration was 0.01 mg/ml and rotor speed was 12 500 r.p.m. Upper panel: Residual from the fit. The estimated GluR1 dimer K_d is ∼270 nM. (B) Similar experimental data and analysis for the GluR2 ATD. Lower panel: absorbance data (red dots) fit to a monomer–dimer model (black line), showing calculated populations of monomer (green) and dimer (blue). The loading protein concentration was 0.01 mg/ml and the rotor speed was 13 000 r.p.m. Upper panel: Residual from the fit. The estimated GluR2 ATD dimer K_d is ∼152 nM. (C) Residuals in absorbance units at 229 nm versus GluR1-ATD concentration (also in absorbance units) from a global fit of three concentrations (0.01, 0.02, 0.05 mg/ml) and three rotor speeds (10 000, 12 500, 17 000 r.p.m.). (D) Residuals in absorbance units at 229 nm versus GluR2–ATD concentration (in absorbance units) from a global fit of three concentrations (0.01, 0.02, 0.04 mg/ml) and three rotor speeds (10 000, 13 000, 18 000 r.p.m.).

Figure 2

Structure of the GluR2-ATD protomer. (A) Ribbon representation of the GluR2 ATD protomer showing the clamshell-like organization of the ATD, domains L1 and L2, α-helices (blue), β-strands (gold) and non α, non β ‘loops' in green. The clamshell-like cleft between domains L1 and L2 is defined by a red star, the amino terminus (N) is at the ‘top', the carboxyl terminus (C), which couples to the S1S2 portion of the receptor, is near the ATD ‘bottom', and two N-acetylglucosamine groups at the well-ordered carbohydrate site (Asn 235) are shown in stick representation. Shown is chain A from the Native 2 crystal structure. (B) View of the protomer following a rotation of ∼90° around a vertical axis. (C) Superposition of the GluR2-ATD protomer (A chain; colours as in panel (A)) and an mGluR1-LBD protomer in an ′open′ conformation (grey; PDB code 1EWK chain B). (D) Similiar superposition as in panel (C), except with the closed, glutamate-bound form of an mGluR1-LBD protomer (grey; PDB code 1EWK; chain A).

Figure 3

Architecture of the GluR2-ATD dimer. (A) View of dimer perpendicular to the molecular two-fold axis. Boxed regions are the crucial L1–L1 and L2–L2 interprotomer interfaces. Helices and strands in a darker shade are either involved in domain–domain interactions (L1–L1: α2/α3; ‘flaps'; L2–L2: α5/β7) or bridge L1 and L2 domains (α8). (B) View of dimer parallel to the molecular two-fold axis looking onto the ‘top' of the L1 domains and into the L1–L1 interface, showing the ‘flaps' and the essential disulphide bonds between the α2 helices and the ‘flaps'.

Figure 4

The L1 ‘Flap'. (A) The L1 ‘flap' folds over the ‘top' of the L1 domain, spans residues Ile298–Trp317, and includes residue Cys309, which makes a disulphide bond to Cys57, as well as Leu310, which forms a hydrophobic contact in the L1–L1 interface. (B) Multiple amino-acid-sequence alignment of the ‘flaps' from AMPA, kainate and NMDA receptors shows that even though the loop varies substantially in length and residue composition from subtype to subtype, the cysteine involved in disulphide bond formation is conserved.

Figure 5

Nature of interprotomer contacts. (A) L1–L1 interface illustrating the proximity of the Cys57–Cys309 disulphide bond and the roles of aromatic–aromatic (Phe50–Phe82) and polar contacts (Thr53, Thr78, Asn54 and Leu310 and the water molecule, HOH63). (B) Multiple amino-acid-sequence alignment of the residues in helices α2 and α3, the two crucial helices comprising the four-helix bundle, L1–L1 interdomain interface. (C) Contacts at the L2–L2 interprotomer interface showing non-polar interactions between the side chains of Leu137, L140 and L144 of helix α5, together with a hydrogen bond between Tyr131 and Gln141, two residues conserved between AMPA and kainate receptors. Here, the molecular two-fold axis is oriented approximately perpendicular to the plane of the page from the vantage point of the ′top′ of the interface, proximal to Leu137. Protomer A is gold, protomer B is blue. The van der Waals surfaces of the leucine side chains are shown as dots. (D) Multiple amino-acid-sequence alignment of residues in helix α5.

Figure 6

Conservation of amino-acid residues and hydrophobic character at the L1–L1 and L2–L2 interfaces. (A) Conservation of amino-acid residues of rat AMPA receptors GluR1–4 mapped onto the protein surface. Red is most conserved and blue is least conserved. (B) Conservation of amino-acid residues of rat AMPA receptors GluR1–4 and rat kainate receptors GluR5–7 mapped onto the solvent-accessible protein surface. Residue colouring is the same as in panel (A). (C) Mapping of residue polarity onto the GluR2-ATD protein surface, coloured accordingly: white, hydrophobic (Ala, Gly, Ile, Leu, Met, Pro and Val); yellow, semipolar (Cys); cyan, polar (His, Asn, Gln, Ser and Thr); red, acidic (Asp and Glu); blue, basic (Lys and Arg); wheat, aromatic (Phe, Tyr and Trp).

Figure 7

Clamshell cleft and hinge. (A) Residues that probably stabilise the partially closed conformation and that are located within the clamshell cleft and near the juncture between the L1 and L2 lobes. (B) Conserved Ala 277, Asp281 and Phe 341 define crucial interactions between helix α9 and the ‘loop' that includes β11 and β12, interactions that also bridge lobes L1 and L2 and modulate possible closed/open cleft conformations.

Figure 8

Role of ATD in iGluR assembly and function. Illustration of two subunits of an iGluR tetramer. Crucial interfaces between ATD subunits are at the L1–L1 and L2–L2 interfaces, emphasised in rectangles. In AMPA and kainate receptors, we suggest that neither of these interfaces nor the ‘clamshell' itself undergoes substantial conformational changes. In NMDA receptors, however, modulatory ligands might induce conformational changes in the clamshell and at the L2–L2 interface (rectangle with hatched lines). These changes, in turn, might be transmitted to the agonist-binding and the ion-channel domains, perturbing the balance between closed and open states.