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Structural mechanism of glutamate receptor activation and desensitization.

Meyerson JR, Kumar J, Chittori S, Rao P, Pierson J, Bartesaghi A, Mayer ML, Subramaniam S - Nature (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, Center for Cancer Research, NCI, NIH, Bethesda, Maryland 20892, USA.

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.

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Antagonist-bound closed state GluA2 density map quality and resolutiona,b, GluA2em 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 GluA2em 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.
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Figure 7: Antagonist-bound closed state GluA2 density map quality and resolutiona,b, GluA2em 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 GluA2em 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.

Mentions: To establish the feasibility of solving iGluR structures with single particle cryo-EM, we first pursued structural studies of fully glycosylated GluA2 with a wild type ATD-LBD linker (referred to as GluA2em) trapped in the closed state with 0.3 mM ZK200775, a high-affinity competitive antagonist14. The 3D structure of GluA2em determined by single particle cryo-EM at a resolution of ~ 10 Å, estimated by the gold standard 0.143 FSC criterion15, demonstrates an overall organization similar to that reported for GluA2cryst (Fig. 1 and Extended Data Fig. 1, 2). The 2-fold symmetric dimer of dimers arrangement of the ATD and LBD, and the domain swap across distal and proximal subunits, are all clearly observed. In the transmembrane domain, similar to the X-ray structure of GluA2cryst6, density for α-helices pre-M1, M1, M3 and M4 are less well-resolved (Extended Data Fig. 2). To obtain a molecular interpretation of the cryo-EM density map, coordinates for two ATD dimers, two LBD dimers and the TM regions derived from GluA2cryst were fit as five independent rigid bodies (Fig. 1). This revealed excellent agreement with the crystal structure, but with an increase in separation between the ATD and LBD, which are ~ 8 Å further apart in GluA2em than in GluA2cryst (Extended Data Fig. 3). We also found a change in angle between LBD dimer pairs, from 139° in GluA2cryst to 144° in GluA2em, and an increase in separation between proximal AC subunits of ~ 5 Å as measured at the top of the LBD in GluA2em. We conclude that deletion of six residues in the ATD-LBD linker, perhaps coupled with crystal packing forces, result in subtle conformational changes, and creation of a buried interface in GluA2cryst that is absent in native AMPA receptors (Fig. 1f).


Structural mechanism of glutamate receptor activation and desensitization.

Meyerson JR, Kumar J, Chittori S, Rao P, Pierson J, Bartesaghi A, Mayer ML, Subramaniam S - Nature (2014)

Antagonist-bound closed state GluA2 density map quality and resolutiona,b, GluA2em 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 GluA2em 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.
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Related In: Results  -  Collection

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Show All Figures
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Figure 7: Antagonist-bound closed state GluA2 density map quality and resolutiona,b, GluA2em 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 GluA2em 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.
Mentions: To establish the feasibility of solving iGluR structures with single particle cryo-EM, we first pursued structural studies of fully glycosylated GluA2 with a wild type ATD-LBD linker (referred to as GluA2em) trapped in the closed state with 0.3 mM ZK200775, a high-affinity competitive antagonist14. The 3D structure of GluA2em determined by single particle cryo-EM at a resolution of ~ 10 Å, estimated by the gold standard 0.143 FSC criterion15, demonstrates an overall organization similar to that reported for GluA2cryst (Fig. 1 and Extended Data Fig. 1, 2). The 2-fold symmetric dimer of dimers arrangement of the ATD and LBD, and the domain swap across distal and proximal subunits, are all clearly observed. In the transmembrane domain, similar to the X-ray structure of GluA2cryst6, density for α-helices pre-M1, M1, M3 and M4 are less well-resolved (Extended Data Fig. 2). To obtain a molecular interpretation of the cryo-EM density map, coordinates for two ATD dimers, two LBD dimers and the TM regions derived from GluA2cryst were fit as five independent rigid bodies (Fig. 1). This revealed excellent agreement with the crystal structure, but with an increase in separation between the ATD and LBD, which are ~ 8 Å further apart in GluA2em than in GluA2cryst (Extended Data Fig. 3). We also found a change in angle between LBD dimer pairs, from 139° in GluA2cryst to 144° in GluA2em, and an increase in separation between proximal AC subunits of ~ 5 Å as measured at the top of the LBD in GluA2em. We conclude that deletion of six residues in the ATD-LBD linker, perhaps coupled with crystal packing forces, result in subtle conformational changes, and creation of a buried interface in GluA2cryst that is absent in native AMPA receptors (Fig. 1f).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, Center for Cancer Research, NCI, NIH, Bethesda, Maryland 20892, USA.

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.

Show MeSH
Related in: MedlinePlus