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Graphical Abstract
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Figure 1. Architecture of the Full-Length Human GluN1-GluN2A Receptor in the High-pH State
(A) Schematic representation of human GluN1EM (cyan) and GluN2AEM (orange) CTD-truncated constructs. Locations of the point mutations (GluN1G612R and GluN2AE656R,E657R) are highlighted by red points and the GluA2 tail fused to the C terminus of GluN2A is shown with a purple line.
(B) A representative fluorescence size-exclusion chromatography of the purified tetrameric GluN1-GluN2AEM receptor. Right panel shows the purified protein on an SDS-PAGE gel stained with Coomassie blue.
(C) Representative 2D class averages of the GluN1-GluN2A receptors at pH 7.8.
(D) Three-dimensional (3D) reconstructed density map of the human GluN1-GluN2A receptor. GluN1 and GluN2A are shown in cyan and orange, respectively, viewed perpendicularly from the overall two-fold y axis of symmetry. Right panels show corresponding coordinate fitting into the density map of the individual GluN1 or GluN2A subunit.
(E) Superimposition of the R1 lobes with the ones in the crystal structure of the GluN1-NTD (Farina et al., 2011) (PDB: 3Q41) or GluN2A-NTD (Romero-Hernandez et al., 2016) (PDB: 5TQ0) showing the opening of the GluN2A-NTD clamshell, and superimposition of the D1 lobes with the ones in the crystal structure of the GluN1-ABD (Yao et al., 2013) (PDB: 4KCC) or GluN2A-ABD (Jespersen et al., 2014) (PDB: 4NF5) showing the closed ABDs upon agonist binding. Opening-closing motions are assessed by the dihedral angles between the upper and lower lobes by connecting the Cα of four residues for the GluN1-NTD (T105, S145, L271, and D227), GluN2A-NTD (E107, Q152, Y281, and E235), GluN1-ABD (I403, S688, V735, and A715) and GluN2A-ABD (L411, S689, V734, and E714). All crystal structures are indicated in purple.
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Figure 2. Cryo-EM Structures of the GluN1-GluN2A Receptor in the Low-pH State
(A) Proton sensitivity of wild-type human GluN1-GluN2A receptors (pH-IC50 = 6.89 ± 0.01, Hill coefficient nH = 1.75, n = 11).
(B) Segmented density map representations of three conformations of GluN1-GluN2A receptors at pH 6.3. GluN1 and GluN2A subunits are shown in pink and green, respectively.
(C) Top-down views of the tetrameric NTDs (top) and ABDs (bottom) of three conformations. The center-of-mass distances (shown as round circles) between the GluN1 and GluN2A subunits are indicated by dashed lines.
(D) Comparisons of the single clamshell domain of the GluN1-NTD, GluN2A-NTD, GluN1-ABD, and GluN2A-ABD in class I with previously reported crystal structures as described in Figure 1E.
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Figure 3. Conformational Mobility Induced by Receptor Protonation
(A) Overall conformational changes of the GluN1-GluN2A heterodimer between the pH 6.3 state (with GluN1 in pink and GluN2A in green) and the pH 7.8 state (gray).
(B) Superimposition of the R1 lobes of individual NTDs of GluN1 or GluN2A. Opening-closing motions are measured as described in Figure 1E. Twisting-untwisting motions are assessed by the dihedral angles between the R1 and R2 lobes by connecting the Cα of M112, G297, E373, and L240 for the GluN2A-NTD.
(C) Superimposition of the R1 lobes of the GluN2A-NTDs shows the motion within the NTD heterodimer. Center-of-mass distances between two R2 lobes of GluN1 and GluN2A are indicated.
(D) Top-down view shows the conformational change of the NTD tetramer by aligning the full-length receptor.
(E and F) Individual GluN1-ABD and GluN2A-ABD show 3.2° and 4.7° rotations, respectively, resulting in the separation of the D1 lobes and gathering together of the D2 lobes along the center y axis. These rotations were defined as the angles of the 3D vectors that wire the center-of-masses of D1 to that of D2 lobes between two pH states.
(G) Top-down view shows the conformational change of the ABD tetramer by aligning the full-length receptor.
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Figure 4. Molecular Dynamics Simulation of NTDs upon Protonation
(A) Distribution of the Cα RMSDs of the tetrameric NTDs.
(B) Upon protonation, RMSD trajectories for each chain (top column) and for the NTD tetramer or two NTD dimers (bottom) were calculated on Cα atoms based on the initial model coordinate (the high-pH model of the human GluN1-GluN2A receptor) within the whole simulation time of 400 ns.
(C) Structural representation of the first essential mode based on analysis of the NTD tetramer trajectory.
(D and E) Dihedral angles measuring the open and closed degrees for GluN1-NTDs (D) and GluN2A-NTDs (E) within the simulation trajectory.
(F and G) Center-of-mass distance changes between the two GluN1-R1 lobes (F) and R2-R2 lobes averaged from the AB and CD heterodimer of GluN1-GluN2A (G) along the whole simulation time.
Right panels in (D)–(G) show the related distribution profiles. See also Figure S4.
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Figure S1. Expression, thermostability, function and cryo-EM reconstruction of human GluN1/GluN2A NMDA receptors. Related to Figure 1 and Figure 2.
(A) Protein expression level of GluN1WT/GluN2AWT, GluN1G612R/GluN2AWT, GluN1WT/GluN2AE656R,E657R, or GluN1G612R/GluN2AE656R,E657R receptors (all with CTD truncated) was measured by fluorescence-detection size-exclusion chromatography (FSEC).
(B) Thermostability test of GluN1WT/GluN2AWT and GluN1G612R/GluN2AE656R,E657R receptors via FSEC experiment. The fluorescence level of GluN1WT/GluN2AWT receptors decreased significantly after heating, while the GluN1G612R/GluN2AE656R,E657R receptors displayed thermostable profiles.
(C) Representative current traces recorded from Xenopus oocytes expressing GluN1WT/GluN2AWT, GluN1G612R/GluN2AWT, GluN1WT/GluN2AE656R,E657R and CTD truncated GluN1WT/GluN2AWT receptors in response to 100 μM glycine and glutamate, respectively. The oocytes were injected with 0.05 ng RNAs for WT and 1.8 ng for the mutant receptors.
(D) A representative micrograph of the glycine/glutamate-bound GluN1/GluN2A receptors at pH 7.8. (E) 3D classification yielded into six classes for GluN1/GluN2A receptors at pH 7.8, whereas the class I containing the largest population was used for further 3D refinement.
(F) Euler angles distribution of assigned to all particles contributing to class I density map of GluN1/GluN2A receptors at pH 7.8.
(G) Fourier shell correlation (FSC) curve of class I density map of GluN1/GluN2A receptors at pH 7.8. The resolution was estimated by cisTEM according to the criterion of gold-standard FSC equaling to 0.143.
(H) A representative micrograph of the glycine/glutamate-bound GluN1/GluN2A receptors at pH 6.3. (I) Representative 2D class average images of the GluN1/GluN2A receptors at pH 6.3.
(J) 3D classification yielded into three classes for GluN1/GluN2A receptors at pH 6.3. (K) FSC curves for the density maps of three classes at low pH calculated by cisTEM.
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Figure S2. Validation of the inter-subunit interfaces by western blot analysis. Related to Figure 1. (A) Surface representation of the human GluN1/GluN2A receptor, with GluN1 in cyan and GluN2A in orange. Right panel: zoom-in views of the cysteine substitutions at the interfaces between GluN1-NTD-R1 and GluN2A-NTD-R2 lobes (Site-I), GluN2A-NTD-R2 and GluN1-ABD-D1 lobes (Site-II), GluN1-ABD-D2 and GluN2A-ABD-D2 lobes (Site-III). The distances between the Cα of putative crosslinking residues are indicated with dash lines.
(B) Western blots data for four pairs of double-cysteine mutants. The heterodimeric bands (about 310 kD) were detected for the GluN1R323C/GluN2AF210C, GluN1N494C/GluN2AT189C, GluN1N494C/GluN2AN193C and GluN1P670C/GluN2AI799C receptors in non-reducing condition (upper panel) and in reducing condition with the presence of β-mercaptoethanol (lower panel). The image in the individual box was from the same blot. All the images were combined together by adjusting the markers to the same level.
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Figure S3. Structural measurements of the GluN1/GluN2 receptors. Related to Figure 1-3.
(A-H) Surface representation of GluN1/GluN2 heterodimeric assemblies in the agonist-bound GluN1/GluN2A receptors at pH 7.8 (A) or at pH 6.3 (B, C, D for class I, II, and III); in the agonist-bound Xenopus (E) or rat (F) GluN1/GluN2B receptors; and the GluN1/GluN2B (G) or GluN1/GluN2A (H) heterodimer in the agonist-bound xenopus triheteromeric NMDA receptor at pH 6.5. Red dots represent the center-of-mass (COM) for individual R1 or R2 lobe on NTDs and D1 or D2 lobe on ABDs. Lines indicate the distance measurements between different COMs.
(I-J) Cartoon representation of different states of GluN2A-NTDs in the cryo-EM structures of human GluN1/GluN2A receptors at the high pH or at the low pH of conformation I, in the crystal structure of di-heteromeric rat GluN1/GluN2A NTDs with the presence of zinc chelator EDTA (PDB:5TQ0) or complexed with the presence of zinc (PDB:5TPW), and in the cryo-EM structure of tri-heteromeric GluN1/GluN2A/GluN2B receptor in agonist-bound state at pH 6.5 (PDB:5UOW). (I) represent the dihedral angles of opening and closure, (J) for the dihedral angels of twisting and untwisting.
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Figure S4. Molecular dynamics simulation of full-length GluN1/GluN2A receptors upon the protonation. Related to Figure 4.
(A) RMSD trajectories for the full-length tetrameric GluN1/GluN2A receptors upon protonation within the whole simulation time of 150 ns.
(B) The open-and-closed dihedral angle measurements for GluN1-NTDs, GluN2A-NTDs, GluN1-ABDs and GluN2A-ABDs along the simulation overall with the increment of time.
(C) The distance change of center-of-masses between the two GluN1-R1 lobes (left panel) and R2-R2 lobes averaged from the two heterodimers of GluN1/GluN2A (right panel) along the whole simulation time.
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