Structural mechanism of glutamate receptor activation and desensitization

Abstract

Ionotropic glutamate receptors are ligand-gated ion channels that mediate excitatory synaptic transmission in the vertebrate brain. To gain a better understanding of how structural changes gate ion flux across the membrane, we trapped rat AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) and kainate receptor subtypes in their major functional states and analysed the resulting structures using cryo-electron microscopy. We show that transition to the active state involves a 'corkscrew' motion of the receptor assembly, driven by closure of the ligand-binding domain. Desensitization is accompanied by disruption of the amino-terminal domain tetramer in AMPA, but not kainate, receptors with a two-fold to four-fold symmetry transition in the ligand-binding domains in both subtypes. The 7.6 Å structure of a desensitized kainate receptor shows how these changes accommodate channel closing. These findings integrate previous physiological, biochemical and structural analyses of glutamate receptors and provide a molecular explanation for key steps in receptor gating.

Extended Data Figure 1. Cryo-electron microscopic imaging of GluA2 with ZK200775

a–c, A series of representative images of GluA2 bound by the competitive antagonist ZK200775 (left panels), with corresponding power spectra and CTF estimates showing signal beyond 8 Å resolution (right panels, solid and dotted lines, respectively). Defocus values are 3.7, 2.7, and 3.0 μm for the three images, respectively. Scale bar is 100 nm.

Extended Data Figure 2. Antagonist-bound closed state GluA2 density map quality and resolution

a,b, GluA2_em antagonist-bound closed state density map with coordinates for ATD dimers, LBD dimers, and the TMD tetramer independently fit to the map. All coordinates were derived from PDB ID: 3KG2. In panel (b) the density map is shown at a higher contour than (a) to highlight the closeness of fit between X-ray coordinates and the density map in the ATD and LBD layers. The density for the ATD-LBD linker region is weaker than that in the rest of the map and is therefore not visible at this threshold. The black bounding box in (b) identifies the M3-helix bundle crossing visible in the density map. c, Visualization of density map to highlight variation in resolution across different regions of the map. The estimated resolution value is color-coded using the scale shown at the bottom edge of the panel. d, Expanded versions of selected regions of map. Roman numerals identify helices 6 and 8, loop 1, and the pre-M1 and M1 helices as indicated in panels (a) and (b). e, A set of plots that include gold-standard FSC plot (black line) for the GluA2_em antagonist-bound closed state density map showing a resolution of 10.4 Å at an FSC value of 0.143, and a plot (red line) of the FSC between the experimentally obtained cryo-EM density map and a map computed from the fitted coordinates, which displays a resolution of 10.6 Å at an FSC value of 0.5, consistent with the gold-standard FSC curve. f, Validation of density map using tilt-pair parameter plot. The spread in orientational assignments around the known goniometer settings is within ~ 25° for > 80 % of the selected particle pairs, with clear clustering observed at the expected location, centered at a distance of 10° from the origin.

Extended Data Figure 3. Assessment of correspondence between GluA2_em and GluA2_cryst

a,b, Density map of antagonist-bound closed state GluA2_em with rigid body fits of GluA2_cryst (PDB ID: 3KG2) reveals separation between the ATD and LBD layers in GluA2_em that is absent in GluA2_cryst due to deletion of six residues in the ATD-LBD linker. In (a), GluA2_cryst fitting was performed using only ATD tetramer coordinates, which reveals a good fit of the ATD layer, but at the expense of a loss of fit of the LBD assembly. Conversely, in (b) fitting was performed using only LBD tetramer coordinates, which reveals a good fit of the LBD layer, but at the expense of a loss of fit of the ATD assembly. The black boxes highlight examples of regions where the mismatches are clearly evident.

Extended Data Figure 4. Open state GluA2 density map quality and resolution

a,b, Density map of glutamate bound GluA2_em in the open state with coordinates for ATD dimers (PDB ID: 3KG2) and glutamate-bound LBD dimers (PDB ID: 1FTJ) fit separately into the map. In panel (b) the density map is shown at a higher contour than (a) to highlight closeness of fit between X-ray domain coordinates and the density map. c, Secondary structural features from ATD chains B/D of the density map corresponding to regions marked in panel (b). Roman numerals identify helices 5, 6, 7 and the ATD lower domain beta sheet. d, Gold-standard FSC plot (black line) for the GluA2_em open state density map showing a map resolution of 12.8 Å at an FSC value of 0.143, and a plot (red line) of the FSC between the experimentally obtained cryo-EM density map and a map computed from the fitted coordinates, which displays a resolution of 12.7 Å at an FSC value of 0.5, consistent with the gold-standard FSC curve.

Extended Data Figure 5. Desensitized state GluA2 density map classes and resolutions

a, Three quisqualate bound GluA2_em desensitized state classes resolved through 3D classification. The maps are the same as those presented in Fig. 2b, but without segmentation to identify the ATD and LBD regions. b, Gold-standard FSC plots for the GluA2_em desensitized state density maps showing resolutions of 21.4 Å, 25.9 Å, and 22.9 Å for classes 1, 2 and 3, respectively at an FSC value of 0.143.

Extended Data Figure 6. Restored open state density map for the GluA2 Quisqualate complex

a, Density map for the GluA2_em open state obtained by addition of the allosteric modulator LY451646 to a suspension of quisqualate-bound, desensitized GluA2. The purpose of the experiment was to test whether structural changes resulting from quisqualate binding to generate the desensitized state could be reversed by addition of an excess of the allosteric modulator LY451646, used to stabilize the open state. The map display shown at left is color-coded to highlight variation in resolution across different regions of the map. b, Density map for the glutamate bound open state obtained by addition of LY451646 30 min prior to agonist, as shown in Figure 2. The map display shown at left is color-coded as in (a) to highlight variation in resolution across different regions of the map. Comparison of the two maps and the fits of ATD and LBD dimers shows that they are essentially identical, establishing the conformational changes that occur with desensitization are reversible and can be modulated by allosteric modulators.

Extended Data Figure 7. Cryo-electron microscopic imaging of GluK2 with 2S,4R-4-methylglutamate and 2D classes

a,b, Representative cryo-EM image of GluK2 bound by the agonist 2S,4R-4-methylglutamate (leftmost panel), with the corresponding image power spectrum and CTF estimate showing signal beyond 8 Å resolution (rightmost panel, solid and dotted lines, respectively). The defocus value of the image is 3.7 μm. Scale bar is 100 nm. c, Two-dimensional classes of desensitized GluK2 particles subjected to single particle analysis. d, Gold-standard FSC plot (black line) for the GluK2 desensitized state density map showing a map resolution of 7.6 Å at an FSC value of 0.143. A plot (red line) of the FSC between the experimentally obtained cryo-EM density map and a map computed from the fitted coordinates, displays a resolution of 7.7 Å at an FSC value of 0.5, consistent with the gold-standard FSC curve.

Extended Data Figure 8. Resolution of the desensitized GluK2 density map

a, GluK2 desensitized state map shown at increasing contour levels from left to right, to better highlight selected secondary structural features. b, Validation of density map using tilt-pair parameter plot. The spread in orientational assignments around the known goniometer settings is within ~ 25° for > 60 % of the selected particle pairs, with clear clustering observed at the expected location, centered at a distance of 10° from the origin. c, Distal (left) and proximal (right) ATD subunits fit with the corresponding X-ray coordinates (PDB ID: 3H6G). d, Proximal (left) and distal (right) LBD subunits fit with the corresponding X-ray coordinates for glutamate-bound GluK2 LBD monomers (PDB ID: 3G3F). The close similarity in density maps for the individual ATD and LBD monomers of distal and proximal domains that are unrelated by computationally imposed C2 symmetry shows that the LBD monomers move largely as rigid bodies and that the structural changes that occur with desensitization can be described adequately by rigid body movements of the ATD and LBD monomers.

Extended Data Figure 9. Comparison between single particle and tomographic reconstructions of desensitized GluK2

a,b, Single particle reconstruction of desensitized GluK2 (a) shown adjacent to the previously reported structure from sub-volume averaging (b). The overall envelope of the two structures are the same, but there is a difference in their length. This difference can be accounted for by considering the effect of the missing wedge on the tomographic structure in (b). c-f, When the two receptor structures are viewed looking down the receptor axis from the extracellular side, ATD layers (c,d) and LBD layers (e,f) can be seen to have the same arrangement. The ATD layer from the single particle structure indicates contact within the ATD tetramer interface (c), and also between LBD monomeric domains (e). As a consequence of the missing wedge in the sub-tomogram average structure, lateral connectivity in the ATD layer (d) and LBD layer (f) is less evident.

Figure 1. GluA2 purification imaging and the antagonist-bound closed state structure

a, FSEC profile for GluA2_em showing a monodisperse profile; the inset shows an SDS PAGE gel for pooled fractions following IMAC purification, after thrombin cleavage to remove the GFP fusion protein, and following preparative SEC. b, Representative power spectrum (solid line) overlaid with the computed contrast transfer function (dashed line) for a cryo-EM image (c), with insets highlighting images of individual GluA2 ZK200775 complexes (scale bar 100 nm). d, Representative 2D class averages from the initial classification of 40,709 projection images. e, Isosurface representation of the GluA2 closed state cryo-EM structure at ~ 10 Å resolution segmented to show distal AC (green and blue), and proximal BD (red and yellow) subunits with GluA2_cryst (PDB ID: 3KG2) coordinates for the ATD, LBD and TM regions fit separately as rigid bodies; the dashed lines highlight putative membrane boundaries. f, Illustration of the region of the LBD layer that is in close contact with the ATD in GluA2_cryst (top panel, cyan shading) that is not observed for GluA2_em (LBD layer of experimental cryo-EM density map, and corresponding fits, are shown in the middle and bottom panels, respectively).

Figure 2. Structural changes accompanying opening of GluA2

a, Representative 2D class averages of GluA2_em in the active state after initial classification of 31,637 projection images. b, GluA2_em active state structure shown in isosurface representation, fitted with ATD dimers (PDB ID: 3KG2) and glutamate-bound LBD dimers (PDB ID: 1FTJ) with the transmembrane region covered by micellar density. c, Density maps for a single subunit showing the visible difference between the antagonist-bound open cleft conformation (top) and the glutamate-bound closed cleft conformation (bottom) of the LBD “clamshell”; the right-hand panel shows the corresponding coordinate fits. d, Ribbon and cylinder diagrams for GluA2 coordinates fit to the closed (magenta) and active (blue) states reveal a ~ 7 Å downward displacement of the ATD in the active state (top), with proximal and distal subunit LBD dimer assemblies viewed perpendicular to (middle) and parallel to (bottom) the membrane. Black dashed lines show the approximate planar interface between subunits in the dimer assembly. e, Isosurface views of LBD tetramer region density maps fit with LBD dimers in closed (left) and active (right) states. Colored dots identify the locations of Cα atoms for Val395 (upper lobe) and Ala665 (lower lobe). f, Movement of the S1-M2 linker (Lys505), M3-S2 linker (Glu634), and S2-M4 linker (Gly771) shows how LBD tetramer movements drive channel opening; arrows show the direction of movement from closed to active states.

Figure 3. Conformational ensemble of desensitized GluA2

a, Representative desensitized state GluA2_em 2D class averages from initial classification of 35,083 projection images. Selected class-averages that illustrate the range of observed conformations are highlighted. b, Segmented isosurface representations of three distinct desensitized state GluA2_em structures, with the ATD and LBD layers identified in blue and orange, respectively. c, Top views of ATD and LBD layers for the three GluA2_em desensitized states (middle columns) flanked by those from the active state (left), and restored active state (right).

Figure 4. GluK2 receptor desensitization

a, GluK2 desensitized state density map at ~ 7.6 Å resolution, segmented and colored to show four receptor subunits, fit with coordinates for GluK2 ATD dimers (PDB ID: 3H6G) and glutamate-bound GluK2 LBD monomers (PDB ID: 3G3F). The GluA2_cryst TM domain was fit as a rigid body. Portions of TM helices where density was only weakly resolved are shown in white. b, Close-up views of selected regions of the density map labeled in (a). c, Top views of density maps for ATD (upper panel) and LBD (lower panel) layers. Colored dots connected by dashed lines identify the locations of His3 and Met382 at the top and base of the ATD, with the progressively smaller parallelograms for Leu402, Lys500 and Ser669 indicating the top, middle and base of the LBD. d, Structural changes in an LBD dimer assembly underlying the transition from the active (blue) to desensitized (magenta) states presented as side (upper panel) and top (lower panel) views. α-helix J, highlighted as a transparent cylinder, and loop 1, marked by an asterisk illustrate the magnitude of LBD rotation with desensitization. Dashed lines show the approximate location of the planar interface between subunits in the domain dimer. e, Movement of the S1-M2 linker (Arg512), M3-S2 linker (Asp638), and S2-M4 linker (Gly772) indicate how LBD tetramer movements drive channel closure; arrows show the direction of movement from active to desensitized states.

Figure 5. Unified view of glutamate receptor gating cycle

a, Schematic summary of global conformational changes highlighting domain movements with channel opening and closure during the receptor gating cycle. The dashed lines over the open state indicate the shortening as a result of the corkscrew rotation that opens the channel. The differences observed between desensitized states of GluK2 and GluA2 are illustrated as variations of a common theme in which the LBD layer shifts to 4-fold symmetry with or without separation of the ATD dimer pairs.