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Distinct structure and gating mechanism in diverse NMDA receptors with GluN2C and GluN2D subunits

Nature Structural & Molecular Biology volume 30, pages 629–639 (2023)Cite this article

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Abstract

N-methyl-d-aspartate (NMDA) receptors are heterotetramers comprising two GluN1 and two alternate GluN2 (N2A-N2D) subunits. Here we report full-length cryo-EM structures of the human N1-N2D di-heterotetramer (di-receptor), rat N1-N2C di-receptor and N1-N2A-N2C tri-heterotetramer (tri-receptor) at a best resolution of 3.0 Å. The bilobate N-terminal domain (NTD) in N2D intrinsically adopts a closed conformation, leading to a compact NTD tetramer in the N1-N2D receptor. Additionally, crosslinking the ligand-binding domain (LBD) of two N1 protomers significantly elevated the channel open probability (Po) in N1-N2D di-receptors. Surprisingly, the N1-N2C di-receptor adopted both symmetric (minor) and asymmetric (major) conformations, the latter further locked by an allosteric potentiator, PYD-106, binding to a pocket between the NTD and LBD in only one N2C protomer. Finally, the N2A and N2C subunits in the N1-N2A-N2C tri-receptor display a conformation close to one protomer in the N1-N2A and N1-N2C di-receptors, respectively. These findings provide a comprehensive structural understanding of diverse function in major NMDA receptor subtypes.

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Fig. 1: Molecular architecture and functional transition of the N1-N2D receptor.
Fig. 2: NTD and LBD function in the N1-N2D receptor.
Fig. 3: Cryo-EM structures and unique allosteric modulation of the N1-N2C di-receptor.
Fig. 4: Structural analysis of the N1-N2A-N2C tri-receptor.
Fig. 5: Comprehensive structural analysis of diverse NMDA receptors.

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Data availability

Cryo-EM density maps and structural coordinates have been deposited in the Electron Microscopy Database and Protein Data Bank under accession codes EMD-33792 and PDB 7YFL for Gly-Glu bound N1a-N2D, EMD-33788 and PDB 7YFF for Gly-CPP bound N1a-N2D, EMD-33795 and PDB 7YFO for crosslinked N1aE698C-N2D, EMD-33798 and PDB 7YFR for non-crosslinked N1aE698C-N2D, EMD-33793 and PDB 7YFM for Gly-Glu bound N1b-N2D, EMD-33789 and PDB 7YFG for Gly-Glu bound N1a-N2C in the asymmetric conformation, EMD-34674 and PDB 8HDK for Gly-Glu bound N1a-N2C in the symmetric conformation, EMD-33790 and PDB 7YFH for PYD-106 bound N1a-GluN2C, and EMD-33791 and PDB 7YFI for Gly-Glu bound N1a-N2A-N2C receptors, respectively. Additional data that support the findings of this study are available from the corresponding author upon request. Source data are provided with this paper.

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Acknowledgements

We thank Y. Kong and L. Pan at the Electron Microscopy Facilities of the Center for Excellence in Brain Science and Technology, Chinese Academy of Sciences for assistance with sample screen. We thank B. Zhu and X. Li at the Center for Biological Imaging (CBI), Institute of Biophysics, Chinese Academy of Sciences, Q. Wang and Y. Zhou at the Electron Microscopy Facility, Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences, and Q. Sun and Y. Wang at the Bio-Electron Microscopy Facility, ShanghaiTech University for their help in cryo-EM data collection. We thank Y. Jia at Fudan University for assistance with single-channel recordings. We acknowledge the technological support of the biological mass spectrometry station of Dalian Coherent Light Source. We are grateful to M. Poo for proofreading. We gratefully acknowledge financial support from the STI2030-Major Project (2022ZD0212700), the National Natural Science Foundation of China (32221003), the Lingang Laboratory (LG 202106-02), the Strategic Priority Research Program of Chinese Academy of Science (XDBS01020000), the Shanghai Municipal Science and Technology Major Project (2018SHZDZX05) and the CAS Youth Interdisciplinary Team to S.Z.

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Author notes

  1. These authors contributed equally: Jilin Zhang, Ming Zhang, Qinrui Wang, Han Wen.

Authors and Affiliations

  1. Institute of Neuroscience, State Key Laboratory of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai, China

    Jilin Zhang, Fenyong Yao, Nan Song, Zengwei Kou & Shujia Zhu

  2. University of Chinese Academy of Sciences, Beijing, China

    Jilin Zhang, Ming Zhang, Yang Li, Fei Guo & Shujia Zhu

  3. Center for Neurological and Psychiatric Research and Drug Discovery, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

    Ming Zhang, Yang Li & Fei Guo

  4. DP Technology, Beijing, China

    Qinrui Wang, Han Wen & Yuhang Wang

  5. CAS Key Laboratory of Separation Sciences for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China

    Zheyi Liu & Fangjun Wang

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Contributions

J.Z. and M.Z. purified and froze the protein, collected and analyzed the cryo-EM data, built atomic models and conducted electrophysiology of the N2D and N2C receptors, respectively. H.W., Q.W. and Y.W. carried out in silico calculations. Z.L. and F.W. performed mass spectrometry. N.S. and Z.K. performed biochemistry assays. F.Y. carried out single-channel recordings. Y.L. and F.G. participated in data collection of N2C receptors, respectively. J.Z., M.Z. and S.Z. wrote the manuscript. S.Z. conceived the project and supervised the research.

Corresponding author

Correspondence to Shujia Zhu.

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Nature Structural & Molecular Biology thanks Albert Lau, Lonnie P Wollmuth and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Katarzyna Ciazynska, in collaboration with the Nature Structural & Molecular Biology team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Protein purification and cryo-EM analysis of Gly-Glu and Gly-CPP bound N1a-N2D receptors.

a, Schematic illustration of CTD-truncated receptor composed of human N1a (in grey) and strep tag fused N2D (in green) subunits. b-d, Coomassie blue gel staining (b) and fluorescence SEC (FSEC) analysis of purified Gly-Glu (c) and Gly-CPP (d) bound N1a-N2D receptors protein. Gel and FSEC analysis were repeated three times. e, f, Cryo-EM data-processing workflow of the Gly-Glu (e) and Gly-CPP (f) bound N1a-N2D receptors. Representative micrographs and 2D average classes are shown. Note that the special 2D averaged class with splayed extracellular domains (highlighted by a red box) was found in both datasets. For both datasets, motion correction, CTF estimation, particle picking and 2D classification were carried out by Relion 3.1.150. Then, 3D classification and 3D refinement were subsequently performed by CryoSPARC51. g, h, Local resolution representation of density map and Fourier shell correlation (FSC) of Gly-Glu (g) and Gly-CPP (h) bound N1a-N2D receptor structures. Maps are colored based on the local resolution estimation by ResMap-1.1.461. Masked (blue) and unmasked (green) FSCs of corresponding maps are both shown, where gold standard FSC = 0.143 was applied for the indication of final resolution (dashed line). i, j, Representation of model fit to map (left two panels) and the angular distribution of particles used in the final reconstruction (right panel).

Source data

Extended Data Fig. 2 Protein purification and cryo-EM analysis of N1b-N2D receptors.

a, Schematic illustration of CTD-truncated receptor composed of human N1b (in grey) and strep tag fused N2D (in green) subunits. b,c, Coomassie blue gel staining and FSEC analysis (repeated three times) of purified proteins of N1b-N2D receptors. d, Cryo-EM data-processing workflow of N1b-N2D receptor datasets processed by Relion 3.1.150. Representative micrographs, 2D average classes, 3D classes and final density maps are shown. e, Local resolution representation of density map and Fourier shell correlation (FSC) of Gly-Glu bound N1b-N2D receptor. Maps are colored based on the local resolution estimation by ResMap-1.1.461. Masked (blue) and unmasked (green) FSCs of corresponding maps are both shown, with gold standard FSC of 0.143 criteria indicated. f, Representation of model fit to map (left two panels) and the angular distribution of particles used in the final reconstruction (right panel).

Source data

Extended Data Fig. 3 Structural, biochemical and electrophysiological analysis of N1a-N2D receptors.

a,b, Conformation comparison of NTDs (a) and protomers (b) between Gly-Glu bound N1-N2D and zinc-bound N1-N2A (PDB:6MMK, ref. 22) di-receptors. Lines, angles and arrows are illustrated as in Fig. 2 legend. c-e, Cartoon representation of the tetrameric interface formed by two N2D-NTDs (c) and two N1-LBDs (d) in Gly-Glu bound N1-N2D receptor, and formed by two N2A-NTDs (d) in Gly-Glu bound N1-N2A receptor (PDB:6MMP, ref. 22), with residues shown in sticks and Cα-Cα distances indicated. f-h, Western blotting analysis on WT and cysteine-substituted N1-N2A (f) and N1-N2D (g, h) receptors (repeated three times). Bands of N1 and N2 momomers, N1-N2 and N2-N2 dimers are indicated. i-j, Representative recording traces and relative MK-801 inhibition on-rate kinetics constants (τon, monoexponential fits) of WT N1-N2D (1.00 ± 0.07, n = 15), N1-N2DS238C(1.01 ± 0.07, n = 12, in i), N1-N2DL822C (0.62 ± 0.08, n = 6, in j), N1E698C-N2D (0.10 ± 0.01, n = 5, in j) and N1E698C-N2DL822C (0.09 ± 0.01, n = 5, in j) receptors. k,l, DTT induced current amplitude changes on WT and mutant receptors. Relative currents (after and before DTT treatment) values, from left to right, are 0.91 ± 0.08 (n = 4), 0.73 ± 0.03 (n = 4), 0.38 ± 0.08 (n = 3) and 0.57 ± 0.13 (n = 5) in k, and 3.56 ± 0.80 (n = 9), 0.65 ± 0.07 (n = 4) and 1.33 ± 0.10 (n = 4) in l. N1* implies the mutant N1C744A, C798A subunit. m, Dose-response curves (fitted by Hill equation) of glycine and glutamate on N1-N2D (Gly EC50 of 0.08 ± 0.01 μM, nH = 1.2 and Glu EC50 of 0.42 ± 0.04 μM, nH = 1.8) and N1E698C-N2D (Gly EC50 of 17.49 ± 0.26 μM, nH = 2 and Glu EC50 of 1.66 ± 0.05 μM, nH = 1.7) receptors. n = 4 oocytes for each group. All data are shown with mean ± SD. For the statistical analysis, P values are determined by two-tailed unpaired Student’s t-test for (i) and by one-way ANOVA followed by the Tukey’s multiple comparison test for (j-l).

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Extended Data Fig. 4 Protein purification, cryo-EM and structural analysis of N1aE698C-N2D receptors.

a-b, Coomassie blue gel staining and FSEC analysis (repeated three times) of purified proteins of N1aE698C-N2D receptors. Bands of N1 and N2D monomer and N1-N1 dimer are indicated. c, Cryo-EM data-processing workflow of N1b-N2D receptor datasets processed by Relion 3.1.150. Representative micrographs, 2D average classes, 3D classes and final density maps are shown. d, Local resolution representation of density map and Fourier shell correlation (FSC) of Gly-Glu bound N1b-N2D receptor. Maps are colored based on the local resolution estimation by ResMap-1.1.461. Masked (blue) and unmasked (green) FSCs of corresponding maps are both shown, with gold standard FSC of 0.143 criteria indicated. e, Representation of model fit to map (left four panels) and the angular distribution of particles used in the final reconstruction (right two panels). f, Structural analysis of top-down viewed tetrameric LBD (left), of side-viewed two N1 (middle) and N2D (right) protomers. Center-of-mass (COM) of each lobe, domain and α-helix E (P670-R673 for N1 and R696-Q699 for N2D) is shown in empty circle. Cα atoms of N1 A652 and N2D A678 in gate are marked in filled circle. The dihedral angles for indicating opening-closure degree of LBD are assessed by connecting the Cα of I403, S688, V735, A715 in N1, and P124, E525, S309, E169 in N2D, respectively. Arrows indicate the conformational changes of Gly-Glu bound N1E698C-2D C-C state compared to the Gly-Glu bound WT receptor. g, Conformational comparison of N2-LBDs in Gly-Glu bound N1a-N2D, Gly-Glu bound N1aE698C-N2D, Gly-Glu bound N1-N2A (PDB:6MMP, ref. 22), Gly-Glu & GNE-6901 bound N1E698C-N2AL794C (PDB:7EOR, ref. 25), Gly-Glu bound WT N1b-N2B (PDB:6WI1, ref. 24), Gly-Glu bound N1bE698C-N2BL795C (PDB:6WHT, ref. 24) receptors. Dash lines indicate COM distances of D1-D1 and D2-D2 lobes.

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Extended Data Fig. 5 Biochemical analysis and Cryo-EM data processing of N1-N2C di-receptors in Gly-Glu bound state.

a, Cartoon representation of N1-N2C receptor, with mRuby-Strep Tag II and 6×His tag placed at the C terminus of N2C and N1 constructs, respectively. b, Coomassie blue gel staining of purified protein of N1-N2C receptors (left panel). FSEC profiles of Gly-Glu bound and Gly-Glu & PYD-106 co-bound N1-N2C receptors (right panel). Gel and FSEC analysis were repeated three times. c, Cryo-EM data-processing flowchart of N1-N2C receptors in complex with Gly-Glu. Representative micrographs, 2D class average images and 3D classification maps are shown. Class 3 of best TMD signal was processed individually to get a map displaying certain signal of TMD. Proportion of particle quantity and NTD top-down view comparison of the asymmetric major, intermediate and symmetric minor classes are shown. Representation of model fit to map, the angular distribution of particles used in the final reconstruction are shown. Masked (blue) and unmasked (green) FSCs of corresponding maps are both shown, with gold standard FSC of 0.143 criteria indicated. Masked (blue) and unmasked (green) FSCs of corresponding maps are both shown, with gold standard FSC of 0.143 criteria indicated. d, Map alignment of asymmetric major (represented by class 3), intermediate (class 8) and minor (class 9) classes. The rotation angle of one NTD heterodimer is indicated. Middle and right panels show the results of three repeats of 200 ns each unrestrained atomistic simulations on both symmetrical and asymmetrical N1A-N2C structures. The histogram and corresponding kernel density estimation are shown. Middle panel shows the NTD-LBD angles of N2C subunits measured throughout the simulations. Right panel shows r.m.s.d. of the least square fitting to the C-alpha atoms, and the first 50 ns of simulations in each repeat were excluded for r.m.s.d. calculation to allow models to fully relax.

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Extended Data Fig. 6 N-linked glycosylation analysis by mass spectrometry.

a, Sequence alignment of the Rat norvegicus N1a, N2A and N2C subunits, highlighted the sites (in purple) detected with N-glycosylation modifications by mass spectrometry. b, Glycans signal (in purple) on EM density maps of N1-N2C (major class) and N1-N2A-N2C receptors are marked. Residue N2CN585 located on the intracellular loop between TM1 and TM2 helices, was detected with N-glycosylation modifications, but not present on the map. c, Statistical chart of site-specified N-glycosylation in N1-N2C di-receptors and N1-N2A-N2C tri-receptors by mass spectrometry. The bar plot summarizes the total count of the different N-glycan compositions, and the pie charts summarize the count distribution of different N-glycosylation types for each N-glycosylated site.

Extended Data Fig. 7 Cryo-EM density maps and structural comparisons of N1-N2C and N1-N2A-N2C receptors.

a, b, Electron density of ligands at the LBD clamshells are shown in red mesh (a). Representative local densities of N2 subunits in Gly-Glu bound (major class) or PYD-106 bound N1-N2C di-receptors, and N1-N2A-N2C tri-receptor structures (b). The models are shown as cartoons and residues are shown as sticks, with N1 coloured in grey, N2A in orange, N2C (chain B) in grey blue and N2C (chain D) in light blue. Agonists Gly and Glu are shown in red sticks. c, Structural comparisons of NTDs and NTD heterodimers within the major class of Gly-Glu bound N1-N2C di-receptor. The r.m.s.d for NTD alignment of N2C (chain B vs chain D) and N1 (chain A vs chain C) are indicated. NTD heterodimers were superimposed using the R1 lobes of N1 with the rotation angles of R2 lobes indicated. d, Structural comparisons of LBD intra-dimer and inter-dimer with the N1-LBDs aligned within the N1-N2C di- (major class, top panel) or N1-N2A-N2C tri-receptors (bottom panel). Rotation angles of N2-LBDs (from N2C to N2A) in the tri-receptor are indicated. Overall, LBDs exhibit pseudo-symmetry in N1-N2C di-receptor and asymmetry in N1-N2A-N2C tri-receptors. e, Arrangement of the NTD tetrameric interface in the asymmetric or symmetric class of N1-N2C di- and N1-N2A-N2C tri-receptors. Three N2C-specific residues (R211, R214 and D220) located at α-helix 5 mediating ionic bond interaction are shaded in red in the sequence alignment of Rat norvegicus N2 subunits at right panel.

Extended Data Fig. 8 Cryo-EM data processing of N1-N2C di-receptors in PYD-106 bound state.

a, Cryo-EM data-processing flowchart of N1-N2C receptors in complex with Gly-Glu & PYD-106. Representative micrographs, 2D class average images and 3D classification maps are shown. To push the resolution of ECD, focused refinement was conducted on the PYD-106 bound N1-N2C receptor, with TMD masked out. For the same purpose, one class of best TMD signal was also processed individually to get a map displaying certain signal of TMD. Masked (blue) and unmasked (green) FSCs of corresponding maps are both shown, with gold standard FSC of 0.143 criteria indicated. b, Representation of model fit to map, the angular distribution of particles used in the final reconstruction are shown.

Extended Data Fig. 9 Molecular mechanism of PYD-106 selectivity on N1-N2C di-receptors.

a, Structural formula of PYD-106 with the only chiral carbon atom in the molecule marked with a red asterisk and the fits of (R)- and (S)-PYD-106 into the EM map which is shown in mesh. Red arrows indicate the unfavorable fitting for (S)-PYD-106. b, Sequence alignment of Rat norvegicus N2A, N2B, N2C and N2D subunits. Red boxes indicate the homologous residues at the bottom of the R2 lobe and the top of the D1 lobe, which directly interact with PYD-106 in Ligplot+ in N2C (chain B). Three key residues that form hydrogen bonds with PYD-106 in N2C (R194, D220 and S472) and their homologous residues are highlighted in red. c, Comparison of the NTD-LBD interfaces of N2 subunits in N1-N2C, N1-N2A (PDB:6MMP, ref. 22), N1-N2B (PDB:7EU8, ref. 26) and N1-N2D di-receptors. The hydrophobicity feature of NTD-LBD interface is highlighted with residues at R2 and D1 lobes.

Extended Data Fig. 10 Purification, biochemical and cryo-EM analysis of the N1-N2A-N2C tri-receptor.

a, Cartoon representation of N1-N2A-N2C tri-receptor, with GFP-6×His tag and GFP-Strep Tag placed at the C terminus of N2A and N2C constructs, respectively. b, Pipeline of two-step affinity chromatography shows that elution of Strep resin was further purified by His resin. Schematic indicates the putative receptor types existed during purification process. Subunit composition at each step was verified by western blotting analysis and the presence of both N2A and N2C subunits was confirmed in the sample after Strep and His affinity purification successively. FSEC profile and Coomassie blue gel staining for the purified tri-receptor protein are shown. Gel and FSEC analysis were repeated three times. c, Flowchart of cryo-EM data-processing for Gly-Glu bound N1-N2A-N2C tri-receptor. The initial model was generated de novo from the selected 2D particles in Relion 3.1.150. One distinctive 3D class (occupied 11.4% particles) showed N1-N2C di-receptor (major class) liked asymmetric features, which was not considered for final 3D refinement. Masked (blue) and unmasked (green) FSCs of corresponding maps are both shown, with gold standard FSC of 0.143 criteria indicated. d, Representation of model fit to map and the angular distribution of particles used in the final reconstruction were shown.

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This video displays the conformational change of the asymmetric N1-N2C di-heteromeric receptor from Gly-Glu to Gly-Glu and PYD-106 bound states, aligned with entire extracellular domains. N1 and N2C subunits are colored in grey and blue, respectively. Upon PYD-106 binding to the N2C of chain B, the relative rotation between NTD and LBD is illustrated.

This video shows the top-down views of asymmetric N1-N2C and N1-N2A (PDB, 6MMP) di-receptors, with N2C (chain B, D) and N2A (chain B, D) colored in blue and orange, respectively. For N1-N2A-N2C tri-receptors, chain B of N2A and chain D of N2C are integrated into the tri-heteromeric receptor assemble.

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Zhang, J., Zhang, M., Wang, Q. et al. Distinct structure and gating mechanism in diverse NMDA receptors with GluN2C and GluN2D subunits. Nat Struct Mol Biol 30, 629–639 (2023). https://doi.org/10.1038/s41594-023-00959-z

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