Architecture and structural dynamics of the heteromeric GluK2/K5 kainate receptor
- Department of Physiology and Biophysics, Weill Cornell Medical College, United States
- Department of Pharmacology and Therapeutics, McGill University, Canada
Figures
Figure 1 with 8 supplements
Structure of the GluK2/K5 heteromer.
(A) Cryo-electron microscopy (cryo-EM) structure of the GluK2/K5em heteromer in a 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)-bound state with GluK2em and GluK5em subunits rendered in green and …
Figure 1—figure supplement 1
Heteromer construct design and structural annotation.
Amino acid sequences for GluK2em and GluK5em cryo-electron microscopy (cryo-EM) expression constructs aligned with wild-type subunit sequences from human (Homo sapiens, hs) and rat (Rattus norvegicus…
Figure 1—figure supplement 2
Heteromer construct illustrations and functional validation.
(A) Illustrations of GluK2em (top, green) and GluK5em (bottom, blue). The GluK2em subunit is annotated to indicate amino terminal domain (ATD), ligand binding domain (LBD), and transmembrane domain …
Figure 1—figure supplement 3
Heteromer biochemistry and cryo-electron microscopy (cryo-EM).
(A and B) Size exclusion chromatography trace for the GluK2/K5em receptor (A) and SDS-PAGE of purified receptor after Coomassie staining (B).
Figure 1—figure supplement 4
Cryo-electron microscopy (cryo-EM) image processing and structure for GluK2/K5em in an apo resting state.
(A) Cryo-EM micrograph of GluK2/K5em-apo. (B) Data processing workflow for GluK2/K5em-apo cryo-EM images. The workflow proceeds from top to bottom. Cryo-EM density maps are color-coded as green or …
Figure 1—figure supplement 5
Analysis of ligand binding domain (LBD) conformations.
(A) Cryo-electron microscopy (cryo-EM) densities for all four LBDs (gray) from the GluK2/K5em-apo cryo-EM map. Each LBD density is fitted with an apo LBD crystal structure (top row, yellow) or a …
Figure 1—figure supplement 6
Cryo-electron microscopy (cryo-EM) image processing workflow for GluK2/K5em with 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX).
(A) Cryo-EM micrograph of GluK2/K5em-CNQX. (B) Data processing workflow for GluK2/K5em-CNQX cryo-EM images. The workflow proceeds from top to bottom. Cryo-EM density maps are color-coded as green or …
Figure 1—figure supplement 7
Resolution determination for GluK2/K5em structures with 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX).
(A, D, and G) Gold-standard Fourier shell correlation (FSC) curves for the unmasked maps (cyan) and masked maps (black). (B, E, and H) Angular distribution for particles in the reconstructions as …
Figure 1—figure supplement 8
Mutually exclusive sites of glycosylation on GluK2 and GluK5 subunits.
(A) The amino terminal domain (ATD) map for GluK2/K5em shown with its structural model. GluK2em and GluK5em subunits are shown in light gray and dark gray, respectively. Selected N-linked glycan …
Figure 2
Analysis of the GluK2/K5 ligand binding domain (LBD) layer.
(A) Extracellular view of LBD layers for (left to right) GluK2/K5em, GluK2, GluN1b/N2B, and GluA1/A2. The LBD layers are extracted from full-length antagonist-bound cryo-electron microscopy …
Figure 3
GluK2/K5 channel structure and symmetry.
(A and B) GluK2/K5em transmembrane domain (TMD) as seen parallel to the membrane (A) and perpendicular to the membrane from the intracellular side (B). Eye icon in (A) gives the perspective …
Figure 4 with 2 supplements
Structure of GluK2/K5 in a desensitized state.
(A) Cryo-electron microscopy (cryo-EM) structure of the GluK2/K5em heteromer in a L-glutamate (L-Glu)-bound state with GluK2em and GluK5em subunits rendered in green and blue, respectively. The …
Figure 4—figure supplement 1
Cryo-electron microscopy (cryo-EM) image processing workflow for GluK2/K5em with L-glutamate (L-Glu).
(A) Cryo-EM micrograph of GluK2/K5em-L-Glu. (B) Data processing workflow for GluK2/K5em-L-Glu cryo-EM images. The workflow proceeds from top to bottom. Cryo-EM density maps are color-coded as green …
Figure 4—figure supplement 2
Resolution determination for GluK2/K5em structures with L-glutamate (L-Glu).
(A, D, and G) Gold-standard Fourier shell correlation (FSC) curves for the unmasked maps (cyan) and masked maps (black). (B, E, and H) Angular distribution for particles in the reconstructions as …
Figure 5 with 1 supplement
The M3-S2 linkers accommodate different ligand binding domain (LBD) arrangements.
(A) GluK2/K5em LBD-transmembrane domain (TMD) assembly, without the amino terminal domain (ATD) layer. Antagonist-bound state (left) annotated with arrows to convey conformational differences with …
Figure 5—figure supplement 1
Cryo-electron microscopy (cryo-EM) density and model for M3 helices, M3-S2 linkers, and E helices.
(A and B) Density map and model for the M3 transmembrane helices, M3-S2 linkers, and E helices of the ligand binding domain (LBD) for GluK2/K5em-6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). The A/C …
Videos
Video 1
Morph for GluK2/K5 ligand binding domain (LBD)-transmembrane domain (TMD) assembly viewed from the side.
The morph presents the conformational differences between antagonist-bound and desensitized states of GluK2/K5em. The LBD and TMD layers are presented without the amino terminal domain (ATD) layer …
Video 2
Morph for GluK2/K5em ligand binding domain (LBD)-transmembrane domain (TMD) assembly viewed from the top.
The morph presents the conformational differences between antagonist-bound and desensitized states of GluK2/K5em. The LBD layer is presented without the amino terminal domain (ATD) or TMD layers, …
Video 3
Morph of M3 and E helices viewed from side.
The movie presents conformational differences in M3 transmembrane helices and E helices of the ligand binding domains (LBDs) between antagonist-bound and desensitized states. The perspective is …
Video 4
Morph of M3 and E helices viewed from top.
The movie presents conformational differences in M3 transmembrane helices and E helices of the ligand binding domains (LBDs) between antagonist-bound and desensitized states. The perspective is …
Tables
Table 1
Summary of kainate receptor (KAR) decay kinetics for wild-type and cryo-electron microscopy (cryo-EM) constructs.
| Desensitization kinetics | ||||||||
|---|---|---|---|---|---|---|---|---|
| Receptor | τ1 ± SEM (ms) | % | τ2 ± SEM (ms) | % | τ3 ± SEM (ms) | % | Weighted τ (ms) | n |
| GluK2 | 6.3 ± 0.3 | 92 | 46 ± 8 | 8 | – | – | 7.5 | 27 |
| GluK2em | 7.7 ± 0.7 | 95 | 41 ± 8 | 5 | – | – | 8.8 | 7 |
| GluK2/K5 | 2.8 ± 0.1 | 92 | 31 ± 6 | 8 | – | – | 4.2 | 26 |
| GluK2/K5em | 2.4 ± 0.1 | 96 | 20 ± 4 | 4 | – | – | 2.9 | 6 |
| Deactivation kinetics | ||||||||
| Receptor | τ1 ± SEM (ms) | % | τ2 ± SEM (ms) | % | τ3 ± SEM (ms) | % | Weighted τ (ms) | n |
| GluK2 | 3.3 ± 0.2 | 97 | 21 ± 3 | 3 | – | – | 3.9 | 28 |
| GluK2em | 4.0 ± 0.4 | 98 | 16 ± 3 | 2 | – | – | 4.2 | 9 |
| GluK2/K5 | 1.2 ± 0.1 | 44 | 10 ± 1 | 21 | 60 ± 2 | 35 | 23.6 | 35 |
| GluK2/K5em | 1.3 ± 0.2 | 60 | 15 ± 5 | 18 | 81 ± 16 | 22 | 18.5 | 6 |
Table 2
Cryo-electron microscopy (cryo-EM) data collection and processing.
| GluK2/K5-apo | GluK2/K5-CNQX (full-length) | GluK2/K5-CNQX (ATD) | GluK2/K5-CNQX (LBD-TMD) | GluK2/K5-L-Glu (full-length) | GluK2/K5-L-Glu (ATD) | GluK2/K5-L-Glu (LBD-TMD) | |
|---|---|---|---|---|---|---|---|
| Magnification | 81,000 | 36,000 | 36,000 | 36,000 | 36,000 | 36,000 | 36,000 |
| Voltage (kV) | 300 | 200 | 200 | 200 | 200 | 200 | 200 |
| Electron exposure (e–/Å2) | 51.23 | 50–53 | 50–53 | 50–53 | 50–53 | 50–53 | 50–53 |
| Defocus (μm) | 1.6 | 0.4–4.8 | 0.4–4.8 | 0.4–4.8 | 0.4–4.8 | 0.4–4.8 | 0.4–4.8 |
| Pixel size (Å) | 1.083 | 1.096 | 1.096 | 1.096 | 1.096 | 1.096 | 1.096 |
| Symmetry imposed | C1 | C1 | C1 | C2 | C1 | C1 | C2 |
| Initial particle images (#) | 1,778,627 | 51,898,826 | 51,898,826 | 51,898,826 | 28,586,529 | 28,586,529 | 28,586,529 |
| Final particle images (#) | 90,027 | 1,021,916 | 540,580 | 184,945 | 573,403 | 241,849 | 140,028 |
| Map resolution (Å) | 7.5 | 5.3 | 3.6 | 4.2 | 5.8 | 3.8 | 4.3 |
| Fourier shell correlation (FSC) threshold | 0.143 | 0.143 | 0.143 | 0.143 | 0.143 | 0.143 | 0.143 |
| Map resolution range (Å) | 6.5–8.0 | 4.0–6.0 | 3.5–4.0 | 4.0–5.0 | 4.0–6.0 | 3.5–4.0 | 4.0–5.0 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Rattus norvegicus) | GRIK2_RAT | Provided by Dr. Janet Fisher (University of South Carolina) | P42260 | |
| Gene (Rattus norvegicus) | GRIK5_RAT | Provided by Dr. Janet Fisher (University of South Carolina) | Q63273 | |
| Cell line (Homo sapiens) | HEK293S GnTI- | ATCC | ATCC, Cat. No. CRL-3022 | |
| Cell line (Homo sapiens) | HEK293T/17 | ATCC | ATCC, Cat. No. CRL-11268 | |
| Cell line (insect) | Sf9 | Expression Systems, courtesy of Dr. Xin-Yun Huang (Weill Cornell Medical College) | Expression Systems, Cat. No. 94–001S | |
| Recombinant DNA reagent | pEZTBM vector | doi: 10.1016/j.str.2016.03.004 | https://www.addgene.org/74099/ | |
| Chemical compound, drug | 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) | Tocris | Tocris, Cat. No. 1045 | |
| Chemical compound, drug | L-Glutamate | Sigma | Sigma, Cat. No. G1251 | |
| Software, algorithm | pClamp 9, Clampfit 10.5 | Molecular Devices, LLC | RRID:SCR_011323 | http://www.moleculardevices.com/products/software/pclamp.html |
| Software, algorithm | OriginPro 2020 | OriginLab | RRID:SCR_014212 | https://www.originlab.com/2020 |
| Software, algorithm | Relion 3.1 | doi:10.7554/eLife.42166 | RRID:SCR_016274 | https://www3.mrc-lmb.cam.ac.uk/relion/index.php/Main_Page |
| Software, algorithm | cryoSPARC 2 | doi:10.1038/nmeth.4169. | RRID:SCR_016501 | https://cryosparc.com/ |
| Software, algorithm | CTFFIND 4.1 | doi:10.1016/j.jsb.2015.08.008 | RRID:SCR_016732 | http://grigoriefflab.janelia.org/ctffind4 |
| Software, algorithm | UCSF Chimera | doi:10.1002/jcc.20084 | RRID:SCR_004097 | http://plato.cgl.ucsf.edu/chimera/ |
| Software, algorithm | UCSF ChimeraX | doi:10.1002/pro.3943 | RRID:SCR_015872 | https://www.cgl.ucsf.edu/chimerax/ |
| Software, algorithm | Bsoft | doi:10.1006/jsbi.2001.4339 | RRID:SCR_016503 | https://lsbr.niams.nih.gov/bsoft/ |
| Software, algorithm | HOLE | doi:10.1016/s0263-7855(97)00009-x | http://www.holeprogram.org | |
| Software, algorithm | COOT 0.9 | doi:10.1107/S0907444910007493 | RRID:SCR_014222 | http://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/ |
| Software, algorithm | Phenix 1.14 | doi:10.1107/S2059798319011471 | RRID:SCR_014224 | https://www.phenix-online.org/ |
| Software, algorithm | GraphPad Prism | GraphPad | RRID:SCR_002798 | http://www.graphpad.com/ |
| Software, algorithm | MolProbity | doi:10.1107/S0907444909042073 | RRID:SCR_014226 | http://molprobity.biochem.duke.edu |
