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Structural insights into binding of therapeutic channel blockers in NMDA receptors

Nature Structural & Molecular Biology volume 29, pages 507–518 (2022)Cite this article

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Abstract

Excitatory signaling mediated by N-methyl-d-aspartate receptor (NMDAR) is critical for brain development and function, as well as for neurological diseases and disorders. Channel blockers of NMDARs are of medical interest owing to their potential for treating depression, Alzheimer’s disease, and epilepsy. However, precise mechanisms underlying binding and channel blockade have remained limited owing to challenges in obtaining high-resolution structures at the binding site within the transmembrane domains. Here, we monitor the binding of three clinically important channel blockers: phencyclidine, ketamine, and memantine in GluN1-2B NMDARs at local resolutions of 2.5–3.5 Å around the binding site using single-particle electron cryo-microscopy, molecular dynamics simulations, and electrophysiology. The channel blockers form different extents of interactions with the pore-lining residues, which control mostly off-speeds but not on-speeds. Our comparative analyses of the three unique NMDAR channel blockers provide a blueprint for developing therapeutic compounds with minimal side effects.

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Fig. 1: Channel blocking of NMDAR by memantine, (S)-(+)-ketamine, and PCP.
Fig. 2: Cryo-EM structure and MD simulations of phencyclidine-bound GluN1a–GluN2B NMDARs.
Fig. 3: Cryo-EM structure and MD simulations of (S)-(+)-ketamine-bound GluN1a-2B NMDARs.
Fig. 4: Cryo-EM structure and MD simulations of memantine-bound GluN1a-2B NMDARs.
Fig. 5: MD simulations of the Thr ring in the presence and absence of channel blockers.
Fig. 6: Effects of interacting residues on association and dissociation kinetics of channel blockers.
Fig. 7: Molecular elements involved in channel blockade.

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Data availability

Cryo-EM density maps for agonist-, (S)-(+)-ketamine-, PCP-, and memantine-bound GluN1a-2B NMDARs have been deposited in the Electron Microscopy Data Bank (EMDB) under accession codes EMD-24946, EMD-24947, EMD-24948, and EMD-24949, respectively, and atomic coordinates have been deposited in the Protein Data Bank (PDB) accession codes 7SAA, 7SAB, 7SAC, and 7SAD, respectively. The MD simulations data have been deposited to Zenodo (https://zenodo.org/record/6380196#.YkNwjSUpCHs). Source data are provided with this paper.

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Acknowledgements

We thank N. Simorowski for technical assistance. D. Thomas and M. Wang are thanked for managing the cryo-EM facility and the computing facility, respectively. T. Heywood is thanked for technical support in usage of HPC facility at Cold Spring Harbor Laboratory (CSHL). We thank M. Mayer for insightful comments on this study. This work was funded by the NIH (NS111745 and MH085926 to H.F.), Robertson funds at CSHL, Doug Fox Alzheimer’s fund, Austin’s purpose, Heartfelt Wing Alzheimer’s fund, and the Gertrude and Louis Feil Family Trust (all to H. F.); and NIH F32MH121061 (to K. M.). The computational work was performed with assistance from an NIH grant (S10OD028632-01). P.C.B thanks the BBSRC for support (BB/M006395/1).

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Author notes

  1. These authors contributed equally: Tsung-Han Chou, Max Epstein, Kevin Michalski.

Authors and Affiliations

  1. W.M. Keck Structural Biology Laboratory, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, NY, USA

    Tsung-Han Chou, Max Epstein, Kevin Michalski, Eve Fine & Hiro Furukawa

  2. Department of Biochemistry, University of Oxford, Oxford, UK

    Max Epstein & Philip C. Biggin

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Contributions

T.-H. C. and E. F. conducted cyro-EM experiments. M. E. conducted MD simulations. K. M. conducted electrophysiology. T.-H. C., M. E., K. M., P. C. B., and H. F. wrote the manuscript.

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Correspondence to Hiro Furukawa.

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Extended data

Extended Data Fig. 1 Single-particle analysis on agonist-bound GluN1a-2B NMDARs.

a, A representative EM micrograph, 2D classes, and the 3D classification/refinement workflow. The scale bar represents 34.9 nm. b, The masked (Blue) and unmasked (Red) Fourier shell correlation (FSC) curves of two half maps (Left) and map vs model (Right). c, Zoom-in views of the gate and the pore-forming region fitted with the molecular model. d, The local resolution estimation calculated by ResMap.

Extended Data Fig. 2 Structural analysis of agonist-bound GluN1a-2B NMDARs.

a, Overall cryo-EM density of the agonist-bound rat GluN1a-2B NMDAR (GluN1a and GluN2B in magenta and forest, respectively). b, EM density of the bound glycine (left) and glutamate (right) at LBD of GluN1a and GluN2B, respectively. c, Zoom-in view of the channel blocker binding cavity at the TMD where there is no density (left). The binding site has three layers, Thr ring (GluN1a-Thr648 GluN2B-Thr647) at the channel gate, the hydrophobic ring (GluN1a-Val644 and GluN1a-Ala645 and GluN2B-Leu643 and GluN2B-Ala644), and the Asn ring (GluN1a-Asn616 and GluN2B-Asn615). d, Domain organization and functional state. The agonist-bound GluN1a-2B NMDAR here has similarity to the non-active1 conformation of GluN1b-2B NMDAR (gray, PDBID: 6WHS) with the similar ATD and LBD orientations and extent of separation between the LBD-TMD linkers of GluN2B (spheres = GluN2B-Gln662).

Extended Data Fig. 3 Single-particle analysis on PCP-bound GluN1a-2B NMDARs.

a, A representative EM micrograph, 2D classes, and the 3D classification/refinement workflow. The scale bar represents 34.9 nm. b, The masked (Blue) and unmasked (Red) Fourier shell correlation (FSC) curves of two half maps (Left) and map vs model (Right). c, Zoom-in views of the gate and the pore-forming region fitted with the molecular model. d, The local resolution estimation calculated by ResMap.

Extended Data Fig. 4 Comparison of channel blocker binding sites.

a-d, Cryo-EM density and fitted models of structures in the absence of a channel blocker (a, apo), PCP (b, orange), S(+)-ketamine (c, cyan), and memantine (d, grey). The residues that are forming the binding site are highlighted as sticks and the densities of the channel blockers are highlighted in green. e-g, Local structural comparison of binding residues between apo and PCP (e), S-(+)-ketamine (f), and memantine (g).

Extended Data Fig. 5 Single-particle analysis on S-(+)-ketamine-bound GluN1a-2B NMDARs.

a, A representative EM micrograph, 2D classes, and the 3D classification/refinement workflow. The scale bar represents 34.9 nm. b, The masked (Blue) and unmasked (Red) Fourier shell correlation (FSC) curves of two half maps (Left) and map vs model (Right). c, Zoom-in views of the gate and the pore-forming region fitted with the molecular model. d, The local resolution estimation calculated by ResMap.

Extended Data Fig. 6 Cryo-EM density at the (S)-(+)-ketamine binding site.

a, Zoom-in views of the cryo-EM density (mesh) fitted with the (S)-(+)-ketamine molecule (cyan sticks) in Pose-3. b, Cryo-EM density of human GluN1a-2A NMDARs (PDBID:7EU7 and EMD:31308, upper panel) complexed to (S)-(+)-ketamine (salmon sticks) and human GluN1a-GluN2B NMDARs (PDBID:7EU8 and EMD:31309, lower panel) complexed with (S)-(+)-ketamine (orange sticks).

Extended Data Fig. 7 Long time-scale MD simulations of (S)-(+)-ketamine-bound GluN1a-2B NMDAR starting from Pose-1.

a, RMSD population density distributions (80 bins per distribution) for three individual Pose-1 1 µs long simulations with runs 1, 2 and 3 each colored blue, orange and green respectively. b, Time series analysis of the (S)-(+)-ketamine Z1 distance expressed as a rolling average (100 frames with a total of 10,000 frames per run) for runs 1 (blue), 2 (orange) and 3 (green). c, Probability density distribution (100 bins) for each independent run of their Z1 (red) and Z2 (green) distances. Dashed lines represent the starting Z1 and Z2 distances of Pose-1.

Extended Data Fig. 8 Single-particle analysis on memantine-bound GluN1a-2B NMDARs.

a, A representative EM micrograph, 2D classes, and the 3D classification/refinement workflow. The scale bar represents 34.9 nm. b, The masked (Blue) and unmasked (Red) Fourier shell correlation (FSC) curves of two half maps (Left) and map vs model (Right). c, Zoom-in views of the gate and the pore-forming region fitted with the molecular model. d, The local resolution estimation calculated by ResMap.

Extended Data Fig. 9 Potency measurement of channel blockers on GluN1a-2B NMDAR.

a-d, Dose-responses of PCP (panel a), S-(+)-ketamine (panel b), memantine (panel c), and Mg2+ (panel d) for wildtype (black circle), GluN1a-V644A (red circle), GluN2B-N615Q (blue triangle), GluN2B-L645A (blue square), and GluN2B-T646S (blue circle) channels recorded on cRNA injected Xenopus oocytes by TEVC. Data points represent the means ± s.d. e, A list of IC50 values, Hill coefficients (nH) calculated based on the dose-response curves. The statistical analysis was done by two-tail Student t-test (*** p < 0.001, ** 0.001 < p < 0.01, * 0.01 < p < 0.05, n.s. not significant. The IC50 values were estimated from independent dose-response recordings from at least four independent oocytes (n).

Source data

Extended Data Fig. 10 Hydrogen-bond network in the Asn ring and extent of hydrophobic interactions.

a,b, Probability density distributions (100 bins) of h-bond distances between all feasible pairs of hydrogen bond doner and acceptor moieties in the Asn ring for the agonist-bound (panel a) and agonist-memantine-bound (panel b) structures. Density distributions for GluN2B-Asn615 (chain B and D) are in green and red. Those for GluN1a-Asn616 (chain A)/GluN2B-Asn615 (chain B and D) and GluN1a-Asn616 (chain C)/GluN2B-Asn615 (chain B and D) are in yellow and blue, respectively. Asterisks denote the interaction pairs for which h-bonds are the main interaction, as suggested by the highest peak at ~2.5 Å. The structures are representative snapshots of collective variables that maximize the total number of possible simultaneous h-bonding interactions. The Asn rings are viewed from the extracellular space where representative h-bonds are depicted as dashed yellow lines. The nitrogen atom h-bond doner variable is represented as the COG between both N+-H atoms. Note that the h-bond network patterns for (S)-(+)-ketamine and PCP are similar to the agonist-bound structure. c, Comparison of binding of PCP pose-1 (orange sticks), memantine (gray sticks), and S-(+)-ketamine pose-2, 3, and 4 (cyan sticks) showing inter-carbon (C-C) interactions within 5 Å (yellow dots) and a hydrogen bond (green dots, arrow).

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Source Data

MD simulation coordinates for (S)-(+)-ketamine (ket_p1.pdb, ket_p2.pdb, ket_p3.pdb, and ket_p4.pdb), PCP (pcp_p1.pdb, and pcp_p2.pdb), and memantine (mematine.pdb)

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Chou, TH., Epstein, M., Michalski, K. et al. Structural insights into binding of therapeutic channel blockers in NMDA receptors. Nat Struct Mol Biol 29, 507–518 (2022). https://doi.org/10.1038/s41594-022-00772-0

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