GluA2 and GluA3 NTDs differ structurally. (A) Left: Topology of an iGluR subunit. The NTD segment is denoted as a green curve and the transmembrane segments as grey columns. Right: Structure of the bipartite GluA2 NTD dimer (PDB 3HSY). The two chains/protomers are coloured green and cyan. Upper and lower lobes (UL, LL) are denoted and their respective interprotomer interfaces are circled. Secondary structural elements contributing to the LL interface are labelled. (B) Structure of the GluA3 NTD (dimer I), with the two protomers coloured red and blue. The UL dimer interface analogous to GluA2 is circled, and the LL interface is shown by a box and an arrow indicating the increased space between the LLs, compared with GluA2. Segments homologous to the GluA2 LL interface segments (from A) are labelled. (C) Lower lobe packing markedly differs between GluA2 and GluA3 NTDs. The lower lobe interface of GluA2 (green) and GluA3 (red) are shown after aligning common secondary structure segments. Note the significantly closer packing of the GluA2 LL interface. Also shown are arginines from GluA3 that project into the interface; this unfavourable electrostatic interaction may contribute to the increased interlobe distance. (D) Sequence conservation in the NTD LL of the AMPA and kainate subfamilies. Different background colours indicate different conservation patterns; for example, conserved sites (columns) within a subfamily are coloured red. Residues that project across the interface are denoted with asterisks (^*). Note the markedly higher conservation of the LL interface within the kainate subfamily. See also Supplementary Figures S1 and S2.

GluA3 NTD crystal structures exhibit different protomerprotomer packings and interfacial contacts. (A) GluA3 crystallizes in three distinct dimeric forms. The dimeric arrangements of each form are shown from above and from the side, with the molecular surfaces of one protomer from dimers I, II and III coloured red, brown and yellow, respectively. (B) Dimers I, II and III are related by rigid-body motions of their protomers. The grey protomers from panel A have been aligned to within 0.5 Å RMSD of each other, whereas the second (coloured) protomer in each structure is left free. The resulting superposition is shown in the inset (the side view from panel A) and turned ∼90°, looking onto the packing surface of each dimer in the main panel. Translational and rotational shifts between interface helices (UL: B and C, LL: E and F) are shown. Note there is a large (∼16 Å) difference between the packing of LL helices in dimer I (red) and II (brown), whereas dimer III (yellow) assumes an intermediate position. The shifts in the UL are indicated for rotation between dimers I and II for helices B and C. The structural difference is more accentuated in the LL, due to intraprotomer structural variabilities. (C) Superposition from B, viewed from the bottom. The two-fold symmetry axis and the plane of the interface are shown as a circle and a dashed line, respectively.

The LLs in GluA2 and GluA3 NTDs exhibit fewer interfacial contacts and larger structural variabilities compared with the ULs. (A) View onto the dimer interface. Atoms making contacts across the dimer interface within 4.5 Å are depicted as spheres on the molecular surface of the respective monomer. Spheres are coloured by number of contacts from blue (1 contact) to red (⩾7 contacts). Local contact density (LD) is also noted for each interface. (B) Structural variations from known GluA2 NTD structures. All GluA2 NTD crystal structures at resolution of 2.5 Å or better (PDB 3HSY, 2WJW, 3H5V) were analysed for different backbone conformations. UL and LL cores from each chain were aligned to a reference chain (3HSY chain B) to within 0.5 Å RMSD and deviations were measured for backbone atoms; these backbone atom deviations are mapped back onto the 3HSY chain B structure and coloured on a logarithmic scale from 0.1 Å (blue) to 10 Å (red). Whereas loops in the UL were largely invariant, specific loops in the LL showed deviations of up to 0.1 Å. Inset: Using the above alignment and superposition algorithm, segments from 3H5V (αF and αG) and 2WJW (αI) exhibiting the most extreme displacements from 3HSY chain B are highlighted in grey. See also Supplementary Figure S4.

Global dynamics of GluA2 and GluA3 NTDs. (A) ANM predicts GluA2 and GluA3 NTDs to adopt classical clamshell motions. One of the dominant modes of motion predicted by ANM simulations for the GluA3 NTD monomer is a classical clamshell opening/closing with a large range of motion. The major deformation is an opening of the cleft (cleft opening angle shown as bars) along the ‘hinge' axis; open (green) and closed (pink) states of GluA3 are shown. (B) Deformations within the dimer assembly are a bit different. The dominant mode is now an anti-correlated motion between the monomers along the axis denoted by the dashed red line, and manifests as a counter-rotation when viewed from the direction indicated by the black arrow. (C) Mobility profile of GluA2, GluA3 and GluK2 NTDs. Residue-specific fluctuations in mode 1 are shown for GluA2 (red), GluA3 (blue) and GluK2 (green; PDB 3H6G) NTDs. The correlation coefficients between the mobility distributions are as follows: 0.82 between GluA2 and GluA3, 0.86 between GluA2 and GluK2 and 0.94 between GluA3 and GluK2. Secondary structural segments that exhibit large fluctuations in the lower lobe (αF, αG and αI) are labelled. Upper and lower lobes are identified as brown and green bars below the x axis. (D) Dimer assemblies also show mobility. A GluA2 NTD dimer is shown with residues coloured by magnitude of the fluctuations from the first 10 modes of GNM, from least (blue) to most mobile (red). Note that the lower lobe is more mobile than the upper lobe, with the putative output region contacting the LBD exhibiting the most mobility. See also Supplementary Figure S5.

High-resolution crystal structure of a GluA2 NTD shows ligand density in the canonical substrate-binding cleft. (A) Residues in GluA2 and GluA3 NTDs critical for collective dynamics. Residues that coordinate hinging motions of the GluA2 and GluA3 NTD dimers are shown as spheres, mapped on the GluA3 dimer I structure. The residues comprise two major groups, based on the two dominant mechanisms of motions: those that coordinate counter-rotations of protomers (UL interfacial residues) and those that coordinate clamshell motions of individual protomers (interlobe hinge residues, i.e., minima from Figure 4B). (B) F_o–F_c electron density maps contoured at 3.0 σ (magenta) and 2.0 σ (blue) show non-protein, non-water molecules in the cleft. Omit difference maps were generated by stripping the cleft of heteroatoms and waters. Putative ligand-coordinating residues are indicated as sticks, with those at analogous positions to ligand-coordinating residues in mGluR1 indicated by red asterisks. Residues identified from normal mode analysis that coordinate collective motions are indicated in blue with analogous positions from GluA3 (from panel B) given in parentheses. All density-coordinating residues are conserved among all AMPA receptors (see Supplementary Figure S6A). The chemical nature of bound ligand is under investigation. See also Supplementary Figure S6.

Allosteric potential of the AMPA receptor NTD. Potential extrinsic NTD modulators may engage different modes of motion. iGluR NTDs have been shown to interact with a variety of extracellular presynaptic, secreted and small-molecule factors. These modulators potentially bind the NTD at multiple sites that can affect the overall conformation in different ways: (1) perturb interprotomer dimer-packing via the sliding interface (purple); (2) regulate intraprotomer conformation via cleft motions (red); or (3) pry apart of pack together the labile lower-lobe interface (blue). Finally, extrinsic factors can affect interdomain conformations by binding to the mobile interdomain linker (4), for example, auto-antibodies in the case of Rasmussen's encephalitis and autoimmune epilepsy (inset, partial epitope shown in brown). These protein–protein interactions, by modulating NTD conformations, have the potential to allosterically control channel function.