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. 2013 Apr 9;110(15):5921-6.
doi: 10.1073/pnas.1217549110. Epub 2013 Mar 25.

Glutamate receptor desensitization is mediated by changes in quaternary structure of the ligand binding domain

David M Schauder  1 , Oleg Kuybeda, Jinjin Zhang, Katherine Klymko, Alberto Bartesaghi, Mario J Borgnia, Mark L Mayer, Sriram Subramaniam
Affiliations

Glutamate receptor desensitization is mediated by changes in quaternary structure of the ligand binding domain

David M Schauder et al. Proc Natl Acad Sci U S A. .

Abstract

Glutamate receptor ion channels are membrane proteins that mediate excitatory synaptic transmission in the central nervous system of vertebrates. Insight into molecular mechanisms underlying glutamate receptor gating is limited by lack of structural information for receptors trapped in different conformational states. Here, we report the use of single-particle cryoelectron tomography to determine the structures, at ∼21 Å resolution, of full-length GluK2 kainate receptors trapped in antagonist-bound resting and agonist-bound desensitized states. The resting state, stabilized by the competitive antagonist LY466195, closely resembles the crystal structure of the AMPA receptor GluA2, with well-resolved proximal and distal subunits exhibiting cross-over between the twofold symmetric amino terminal domain and a twofold symmetric ligand binding domain (LBD) dimer of dimers assembly. In the desensitized state, the LBD undergoes a major rearrangement, resulting in a separation of the four subunits by ∼25 Å. However, the amino terminal domain, transmembrane, and cytoplasmic regions of the receptor have similar conformations in the resting and desensitized states. The LBD rearrangement was not anticipated in prior models based on crystal structures for soluble LBD dimer assemblies, and we speculate that subunit separation allows a better match to the fourfold symmetric ion channel domain. From fits of the amino terminal domain and LBD domains into the density map of the desensitized state we have derived a structural model for differences in quaternary conformation between the resting and desensitized states.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
GluK2 preparations used for structural analysis. (A) Tryptophan fluorescence size-exclusion chromatograms for GluK2 reveals elution as monodisperse tetramers in the apo state, and in the presence of either 1 mM 2S,4R-4-methylglutamate or 1 mM LY466195; the inset shows a Coomassie blue-stained SDS polyacrylamide gel during purification by immobilized metal affinity chromatography (IMAC), thrombin digestion (Thr), and size-exclusion chromatography (SEC), with bands at the expected molecular weight (MW) of 128.4, 99.5, and 28.9 kDa for the GluK2–EGFP fusion protein and the cleaved products. (B and C) Slices from tomograms obtained using plunge-frozen GluK2 specimens in the presence of LY466195 (B) or 2S,4R-4-methylglutamate (C). The red circles highlight individual GluK2 complexes. (Scale bar, 50 nm.)
Fig. 2.
Fig. 2.
Density maps for GluK2 resting and desensitized states. (A and B) Isosurface representations in the resting (A) and desensitized (B) states obtained by cryoelectron tomography with subvolume averaging. (C and D) Absolute density representations clearly reveal differences between the two conformations; in each panel, the images in the upper row show four projections of the front view of the maps shown in A and B, spaced by successive rotations around the long axis by 15°; the bottom rows show five sections through the maps perpendicular to the long axis, at the locations indicated by the red arrows between A and B; the numbered arrows correspond to the sections shown in C and D, with approximate locations as follows: (1) middle of the transmembrane region, (2) membrane-proximal region on the extracellular side, (3) lower lobe of the ligand-binding domain (LBD), (4) upper lobe of the LBD, and (5) central region of the ATD.
Fig. 3.
Fig. 3.
Molecular architecture of GluK2 complexed to LY466195 (resting state) fit with GluA2cryst (3KG2) coordinates. (A) Top and (C) side views illustrating the locations of the ATD and LBD relative to the lipid bilayer, and the cross-over between the ATD and LBD; the red arrow shows where six amino acids and two glycosylation sites were deleted in GluA2cryst (4). (B) Density map obtained by filtering coordinates for GluA2cryst to a resolution of 21 Å, superimposed on the GluK2 resting-state map.
Fig. 4.
Fig. 4.
Separation of LBD protomers in the GluK2 desensitized state. Slices through the LBD (Upper) and ATD (Lower) regions in resting- (A) and desensitized-state (B) density maps shown as isosurface representations, with fits of 3KG2 coordinates. There is a large rearrangement in the LBD layer on desensitization, which results in separation of the four GluK2 protomers with only a small rotation of the ATD layer. Fits were obtained using automated procedures, except for the LBD desensitized state, which was performed manually.
Fig. 5.
Fig. 5.
Quaternary structures of resting and desensitized states. (A and B) Density maps rendered to show GluK2 subunits in the tetrameric assembly. The desensitized state displays a clear separation of LBD protomers with a small twist of ATD dimers compared with the resting state. (C and D). Sections through the LBD, illustrating the dimer-of-dimers assembly in the resting state and domain separation in the desensitized state, accompanied by large rotations of the proximal (P), but not distal (D), subunit pairs. (E and F), Top view through the density maps of the resting (E) and desensitized (F) states where the LBD regions are approximated with molecular envelopes at 20 Å resolution to illustrate the conformational change. In the resting state, the entire LBD tetramer was fit automatically using UCSF Chimera, whereas for the desensitized state the four monomers were placed manually in the map in the absence of strong structural constraints to produce reliable automated fits.
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