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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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GluA2 purification imaging and the antagonist-bound closed state structurea, FSEC profile for GluA2em 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 GluA2cryst (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 GluA2cryst (top panel, cyan shading) that is not observed for GluA2em (LBD layer of experimental cryo-EM density map, and corresponding fits, are shown in the middle and bottom panels, respectively).
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Figure 1: GluA2 purification imaging and the antagonist-bound closed state structurea, FSEC profile for GluA2em 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 GluA2cryst (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 GluA2cryst (top panel, cyan shading) that is not observed for GluA2em (LBD layer of experimental cryo-EM density map, and corresponding fits, are shown in the middle and bottom panels, respectively).

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)

GluA2 purification imaging and the antagonist-bound closed state structurea, FSEC profile for GluA2em 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 GluA2cryst (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 GluA2cryst (top panel, cyan shading) that is not observed for GluA2em (LBD layer of experimental cryo-EM density map, and corresponding fits, are shown in the middle and bottom panels, respectively).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4199900&req=5

Figure 1: GluA2 purification imaging and the antagonist-bound closed state structurea, FSEC profile for GluA2em 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 GluA2cryst (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 GluA2cryst (top panel, cyan shading) that is not observed for GluA2em (LBD layer of experimental cryo-EM density map, and corresponding fits, are shown in the middle and bottom panels, respectively).
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