Glutamate receptors are ligand-gated tetrameric ion channels that mediate synaptic transmission in the central nervous system. They are instrumental in vertebrate cognition and their dysfunction underlies diverse diseases1,2. In both the resting and desensitized states of AMPA and kainate receptor subtypes, the ion channels are closed, whereas the ligand-binding domains, which are physically coupled to the channels, adopt markedly different conformations3,4,5,6. Without an atomic model for the desensitized state, it is not possible to address a central problem in receptor gating: how the resting and desensitized receptor states both display closed ion channels, although they have major differences in the quaternary structure of the ligand-binding domain. Here, by determining the structure of the kainate receptor GluK2 subtype in its desensitized state by cryo-electron microscopy (cryo-EM) at 3.8 Å resolution, we show that desensitization is characterized by the establishment of a ring-like structure in the ligand-binding domain layer of the receptor. Formation of this ‘desensitization ring’ is mediated by staggered helix contacts between adjacent subunits, which leads to a pseudo-four-fold symmetric arrangement of the ligand-binding domains, illustrating subtle changes in symmetry that are important for the gating mechanism. Disruption of the desensitization ring is probably the key switch that enables restoration of the receptor to its resting state, thereby completing the gating cycle.
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Nature Methods Open Access 01 August 2022
Allosteric coupling of sub-millisecond clamshell motions in ionotropic glutamate receptor ligand-binding domains
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Nature Communications Open Access 05 August 2021
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Cryo-EM density maps for GluK2EM with 2s,4R-4-methylglutamate, and GluK2EM with LY466195, have been deposited in the Electron Microscopy Data Bank under accession codes EMD- 8289 and EMD- 8290, respectively. Atomic coordinates for GluK2EM with 2s,4R-4-methylglutamate, GluK2EM with LY466195, GluK2EM LBD with 2s,4R-4-methylglutamate, and GluK2EM LBD with LY466195, have been deposited in the Protein Data Bank under accession codes, 5KUF, 5KUH, 5CMM and 5CMK, respectively.
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This research was supported by the intramural programs of the NCI, and NICHD, NIH, the IATAP program at NIH, and the NIH-FEI Living Laboratory for Structural Biology. Synchrotron diffraction data was collected at Southeast Regional Collaborative Access Team (SER-CAT) 22-ID beamline at the Advanced Photon Source (APS), Argonne National Laboratory. Use of APS was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract number W-31-109-Eng-38. We thank D. Bleakman for the gift of LY466195, C. Glasser for preparing DNA constructs, and F. DiMaio and colleagues for assistance with use of the program Rosetta. We also thank V. Falconieri for preparing the schematic illustration panel. This study used the capabilities of the HPC Biowulf cluster at NIH, Bethesda, MD (http://hpc.nih.gov).
The authors declare no competing financial interests.
Reviewer Information Nature thanks L. Wollmuth and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
a, b, Representative cryo-EM image of GluK2EM solubilized in DDM–CHS and bound by 2S,4R-4-methylglutamate (a), with the corresponding image power spectrum and CTF estimate showing signal beyond 3 Å resolution (b; solid and dotted lines, respectively). The defocus value for the image is 1.5 μm. Particles in a are highlighted with circles, and image binning at 4× and uniform level adjustments were used to make particles apparent. Scale bar, 500 Å. c, Subset of selected two-dimensional class averages. d, FSC curve with reported resolution of 3.8 Å at the 0.143 crossing. e, Structure of agonist-bound GluK2EM, coloured according to local resolution, shown at three progressively increasing contours.
a, b, Cryo-EM density for resolved S1–M1 (a) and M3–S2 (b) linkers and transmembrane helices, displayed with Cα trace of the atomic model. c, Representative sites with densities for complex glycans at Asn244, Asn347 and Asn399.
a, Cryo-EM density map for the reconstruction of agonist-bound GluK2 without imposition of computational symmetry, and coloured according to local resolution. b, FSC curve with reported resolution of 4.4 Å at the 0.143 crossing. c, Segmentation of individual GluK2 chains of the asymmetric reconstruction. The density map segmentation is shown fitted with a trace representation of the model displayed in Fig. 1b.
Extended Data Figure 4 Inhibition of GluK2EM by LY466195 and LBD crystal structures for agonist and antagonist complexes.
a, Crystal structure for the GluK2EM-isolated LBD dimer assembly complex with 2S,4R-4-methylglutamate. The upper and lower lobes for the two subunits are coloured orange and pale yellow, and teal and pale cyan, respectively; the dashed line indicates the separation of the lower lobes measured as the distance between the Cα positions of Ile637. b, Crystal structure for the GluK2EM-isolated LBD dimer assembly complex with LY466195 illustrating the large decrease in separation of the lower lobes compared to the agonist complex. Colouring is the same as in a. c, Responses to 100 μM glutamate recorded under two electrode voltage clamp for GluK2EM (top) and wild-type GluK2 (bottom); the initial response to glutamate recorded after prior application of 300 nM LY466195 showed nearly complete block for GluK2EM, with no change in amplitude for wild type. d, Concentration dependence for inhibition of GluK2EM by LY466195 yielded an IC50 of 30 nM; data points show mean ± s.e.m. of 4–7 observations per concentration.
a, b, Representative cryo-EM image of GluK2EM solubilized in DDM-CHS and bound by LY466195 (a), with the corresponding image power spectrum and CTF estimate showing signal beyond 6 Å resolution (b, solid and dotted lines, respectively). The defocus value for the image is 2.7 μm. Particles in a are highlighted with circles, and image binning at 4× and uniform level adjustments were used to make particles apparent. Scale bar, 500 Å. c, Subset of selected two-dimensional class averages. d, Cryo-EM density map for GluA2 bound to ZK200775 (ref. 4) (left), density map for GluK2EM bound to LY466195 (middle) and its corresponding molecular model built from ATD and LBD dimers (right). e, FSC curve with reported resolution of 11.6 Å at 0.143 crossing.
a, Desensitized GluK2 shown in surface representation with ATD–LBD interfaces highlighted. b, Top-down view of LBD layer shown with perspective indicated by eye icon in a. c, Underside of the ATD layer as viewed after peeling away from LBD layer. Dashed lines in a highlight where the layers are separated. In all panels, interfaces on chain A and C are in green and blue, respectively. d, Table with residues that mediate ATD–LBD interaction. e, f, Cartoon representation of LBD and ATD layers from same views as in b and c, with interface residues coloured to correspond with table in d.
a–d, Rate of recovery from desensitization measured using twin pulse applications of 10 mM glutamate (data points show mean ± s.d.; fits are shown in red). a, Wild-type GluK2 fit with a single exponential. b, D672R fit with the sum of two exponentials with the response for wild type shown as a black line; c, S669R D672R double mutant fit with the sum of two exponentials with the response for wild type shown as a black line. d, Time to 50% recovery in seconds. e–h, Top views of the GluK2 desensitization ring with residues found to influence recovery kinetics when mutated. Each panel shows the wild-type residue Cα position as a sphere. Positions for the S669R and D672R mutations from the present study are shown in e and f, respectively. The A676T and S679R positions found in previous studies20,21 are in g and h, respectively.
The transitions between resting, activated, and desensitized states are shown here. The resting and desensitized state models were built as described in the methods section. The activated state model was built using the GluA2EM activated state and GluA2cryst structures as guides to reveal conformational changes in the ATD and LBD layers, with the GluA2cryst ion channel used as a reference for positioning in the vertical plane. (MOV 13322 kb)
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Meyerson, J., Chittori, S., Merk, A. et al. Structural basis of kainate subtype glutamate receptor desensitization. Nature 537, 567–571 (2016). https://doi.org/10.1038/nature19352
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