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Glycine receptor mechanism elucidated by electron cryo-microscopy

Naturevolume 526, pages224–229 (2015)Cite this article

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Abstract

The strychnine-sensitive glycine receptor (GlyR) mediates inhibitory synaptic transmission in the spinal cord and brainstem and is linked to neurological disorders, including autism and hyperekplexia. Understanding of molecular mechanisms and pharmacology of glycine receptors has been hindered by a lack of high-resolution structures. Here we report electron cryo-microscopy structures of the zebrafish α1 GlyR with strychnine, glycine, or glycine and ivermectin (glycine/ivermectin). Strychnine arrests the receptor in an antagonist-bound closed ion channel state, glycine stabilizes the receptor in an agonist-bound open channel state, and the glycine/ivermectin complex adopts a potentially desensitized or partially open state. Relative to the glycine-bound state, strychnine expands the agonist-binding pocket via outward movement of the C loop, promotes rearrangement of the extracellular and transmembrane domain ‘wrist’ interface, and leads to rotation of the transmembrane domain towards the pore axis, occluding the ion conduction pathway. These structures illuminate the GlyR mechanism and define a rubric to interpret structures of Cys-loop receptors.

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Figure 1: Glycine receptor architecture.
Figure 2: The ion channel.
Figure 3: Strychnine and ivermectin bind at subunit interfaces.
Figure 4: Conformational changes within an individual subunit.
Figure 5: Conformational differences at the subunit–subunit interface between agonist- and antagonist-bound states.
Figure 6: Overall conformational changes of the TMD.

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Electron Microscopy Data Bank

Protein Data Bank

Data deposits

Three three-dimensional cryo-EM density maps and coordinates of α1 glycine receptors in strychnine-bound, glycine-bound and glycine/ivermectin-bound forms have been deposited in the Electron Microscopy Data Bank under the accession numbers EMD-6344, EMD-6345, and EMD-6346 and deposited in the RCSB Protein Data Bank under the accession codes3JAD,3JAE, and3JAF.

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Acknowledgements

We are grateful to Z. H. Yu, N. Grigorieff, J. Cruz and C. Hong (Janelia Campus), C. Arthur (FEI) and M. Braunfeld (UCSF) for assistance with microscope operation, data collection and for comments, and to R. Stites, M. Hakanson and A. Trzynka (OHSU) for computational support. We acknowledge the support of R. Goodman and J. Gray. Microscopy at Oregon Health & Science University (OHSU) was performed at the Multiscale Microscopy Core (MMC) with technical support from the OHSU-FEI Living Lab, Intel and the OHSU Center for Spatial Systems Biomedicine (OCSSB). We thank L. Vaskalis for help with illustrations and H. Owen for proofreading. R. Hibbs is gratefully acknowledged for pre-screening the GlyR constructs and D. P. Claxton for optimizing the constructs. We thank Gouaux and Baconguis laboratory members for discussions. This work was supported by the National Institute of Health (E.G). E.G. is an investigator with the Howard Hughes Medical Institute.

Author information

Author notes

  1. Juan Du and Wei Lü: These authors contributed equally to this work.

Authors and Affiliations

  1. Vollum Institute, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, 97239, Oregon, USA

    Juan Du, Wei Lü & Eric Gouaux

  2. Department of Biochemistry and Biophysics, University of California San Francisco, 600 16th Street, San Francisco, 94158, California, USA

    Shenping Wu & Yifan Cheng

  3. Howard Hughes Medical Institute, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, 97239, Oregon, USA

    Eric Gouaux

Authors
  1. Juan Du

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  2. Wei Lü

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  3. Shenping Wu

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  5. Eric Gouaux

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Contributions

J.D., W.L. and E.G. designed the project, J.D. and W.L. performed sample preparation, cryo-EM data collection and data analysis, J.D., W.L. and E.G. wrote the manuscript, S.W. and Y.C. assisted in cryo-EM experiments at UCSF and participated in discussion and editing of the manuscript.

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Correspondence toEric Gouaux.

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Extended data figures and tables

Extended Data Figure 1 Three-dimensional reconstruction of strychnine-bound GlyR.

a, A representative micrograph (out of 1,829 micrographs) of strychnine-bound GlyR in vitreous ice.b,c, Angular distribution of particle projections (b), and selected 2D classes (c) are shown. Inc, the radius of the sphere is proportional to the number of particles assigned to it. The plot is drawn with respect to the 3D reconstruction shown in the centre, taking the C5 symmetry of the receptor into account.d, Selected ‘slice’ views of the final reconstruction along the pore axis. The slice numbers are indicated, starting from the intracellular side.e, FSC curves for the density maps before (red) and after (black) post-processing in RELION. The FSC curve between the refined atomic model and the final reconstruction map is shown in green.f, FSC curves for cross-validation: model versus summed map (full data set, green), model versus half map 1 (used in test refinement, orange) and model versus half map 2 (not used in test refinement, purple).g, Unfiltered and unsharpened 3D density map coloured according to local resolution estimated using RESMAP65.h, Real-space correlation between atomic model and density map calculated using PHENIX.

Extended Data Figure 2 3D reconstruction of glycine-bound GlyR.

a, A representative micrograph (out of 1,460 migrographs) of glycine-bound GlyR in vitreous ice.b,c, Angular distribution of particle projections (b) and selected 2D classes (c) are shown. Inc, the radius of the sphere is proportional to the number of particles assigned to it. The plot is drawn with respect to the 3D reconstruction shown in the centre, taking the C5 symmetry of the receptor into account.d, Selected ‘slice’ views of the final reconstruction along the pore axis. The slice numbers are indicated, starting from the intracellular side.e, FSC curves for the density maps before (red) and after (black) post-processing in RELION. The FSC curve between the refined atomic model and the final reconstruction map is shown in green.f, FSC curves for cross-validation: model versus summed map (full data set, green), model versus half map 1 (used in test refinement, orange) and model versus half map 2 (not used in test refinement, purple).g, Unfiltered and unsharpened 3D density map coloured according to local resolution estimated using RESMAP.h, Real-space correlation between atomic model and density map calculated using PHENIX.

Extended Data Figure 3 3D reconstruction of glycine/ivermectin-bound GlyR.

a, A representative micrograph (out of 2,489 micrographs) of glycine/ivermectin-bound GlyR in vitreous ice.b,c, Angular distribution of particle projections (b) and selected 2D classes (c) are shown.c, The radius of the sphere is proportional to the number of particles assigned to it. The plot is drawn with respect to the 3D reconstruction shown in the centre, taking the C5 symmetry of the receptor into account.d, Selected ‘slice’ views of the final reconstruction along the pore axis. The slice numbers are indicated, starting from the intracellular side.e, FSC curves for the density maps before (red) and after (black) post-processing in RELION. The FSC curve between the refined atomic model and the final reconstruction map is shown in green.f, FSC curves for cross-validation: model versus summed map (full data set, green), model versus half map 1 (used in test refinement, orange) and model versus half map 2 (not used in test refinement, purple).g, Unfiltered and unsharpened 3D density map coloured according to local resolution estimated using RESMAP.h, Real-space correlation between atomic model and density map calculated using PHENIX.

Extended Data Figure 4 Representative densities of the three reconstructions of GlyR. Densities are sharpened using RELION unless indicated otherwise.

The densities in each panel are for the strychnine-, glycine/ivermectin-, glycine-, and unsharpened glycine-bound states, respectively, from left to right.a, Representative densities of the β-sheets in ECD, contoured at 8σ.b, Densities of Cys loop and the M2–M3 loop, contoured at 7σ.c, Densities of helices M1 and M2, contoured at 7σ.d, Densities of M3 and M4, contoured at 7σ.e, Densities of −2′Pro, contoured at 7σ except for the glycine-bound state (6.5σ).f, Densities of 9′Leu, contoured at 6.0σ except for the glycine-bound state (5.0σ).

Extended Data Figure 5 A single subunit of glycine/ivermectin bound GlyR.

a–c, Viewed in parallel to the membrane plane, with secondary structure elements labelled.b, The domain arrangement resembles an upright forearm, clad with a mitten, consisting of thumb (C loop), palm (β-strands of ECD) and ligament (ECD–TMD interface).

Extended Data Figure 6 Comparison of ivermectin-binding site in GlyR (red) and GluCl (green).

a, Viewed in parallel to the membrane.b, Viewed from the extracellular side. The (+)-subunits are shown in darker colours. The residue corresponding to Arg287, which forms a hydrogen bond with the ivermectin in GlyR, is an asparagine (Asn264) in GluCl. The corresponding residue of Val296 in the M2–M3 loop of GlyR is an isoleucine (Ile273) in GluCl, whose larger side chain prevents the upper tip of ivermectin from approaching and interacting with the main chain oxygen atom of Ser721 in the M2–M3 loop (Ser294 in GlyR). The Gly237 in the M1 and Ala304 in the M3 of GlyR are Ser217 and Gly281 in GluCl, respectively. Such differences on side chains weaken or strengthen the interaction of ivermectin with M3 or M1 in GlyR, respectively, in comparison to that in GluCl.

Extended Data Figure 7 Comparison of GlyR with other Cys-loop receptors.

a, The two restriction sites, viewed from the cytoplasmic side. The Cα of −2′Pro equivalents (cyan) and 9′Leu equivalents (magenta) are shown as spheres. Distances between adjacent Cα atoms are labelled.b, Plot of the vector connecting the −2′ProCα equivalent and 9′LeuCα equivalent, with −2ProCα equivalent as the origin, the tilt angle θ and the rotation angle ϕ relative to the pore axis. The ϕ of strychnine-bound GlyR is arbitrarily set to zero.c, Pore radii as a function of distance along the pore axis, calculated using the program HOLE, where the Cα position of 0′Arg is set to zero.d, Table showing parameters of the vector connecting the −2′ProCα equivalent and 9′LeuCα equivalent, wherer is the distance from −2′ProCα equivalent to 9′LeuCα equivalent,RestrP andRestrL are the pore radii at −2′Pro equivalent and 9′Leu equivalent, respectively. ThedC loop is the distance between Cα of Thr220 equivalent and Leu143 equivalent, representing the opening of the C loop shown ine.e, Comparison of ligand-binding pockets. The side chains of marker residues are shown in stick format.

Extended Data Figure 8 Superimposition of TMD in three GlyR structures using main chain atoms of residues Met236–Lys362.

a, Between strychnine- and glycine–GlyR.b, Strychnine- and glycine/ivermectin–GlyR.c, Glycine- and glycine/ivermectin–GlyR. The M2–M3 loop, residues Ser289–Ala298, is excluded from the comparison. The root mean square deviations (r.m.s.d.) are 0.9, 0.9, and 0.7 Å, respectively, suggesting that the movement of the TMD is rigid-body-like. Most differences are located in the termini of transmembrane helices, which are either close to the M2–M3 loop, or close to the intracellular gate −2′Pro.

Extended Data Figure 9 Positions of residues in which mutations are associated with human startle disease.

Residues that likely interact with disease‐causing residues are labelled in italics.a, The strychnine–GlyR model is used to show residues in which mutations cause spontaneous activation. The mutation of Gln242 in M1 to glutamate may enhance its electrostatic attraction to Arg287 in M2 of the adjacent subunit and tilt the upper part of M2 away from pore axis, resulting in a constitutively open channel. Alternatively, the mutation Val296Met in M2–M3 loop may cause steric collision with Ile241 in M1 of the adjacent subunit, and prevent Ser294 from interacting to the N‐cap formed by pre‐M1, M1 and the β8–β9 loop, thereby destabilizing the closed conformation.b, The glycine/ivermectin–GlyR model is used to show residues in the ECD–TMD interface whose mutations reduce sensitivity to glycine and single channel conductance. The mutation of Arg234 in pre‐M1 to glutamine may disturb its electrostatic interaction with Asp164 in the Cys loop. Similarly, the mutation of Tyr295 in the M2–M3 loop to cysteine or serine may disturb its interaction with the main chain nitrogen atom of Leu158 in the Cys loop. In both cases, the signal induced by agonist binding may be blocked. The mutation Lys292Glu in the M2–M3 loop possibly affects the cooperative interaction between two adjacent subunits by altering the van der Waals contacts between Lys292 and Tyr238.c, The glycine–GlyR model is used to show residues in M2 in which mutations reduce sensitivity to glycine and diminish single channel conductance. These mutations may directly influence the pore properties by modifying the interactions with adjacent residues, for instance, between Gln282His and Pro246, and between Arg287Gln/Leu and Gln242.

Extended Data Table 1 Statistics of 3D reconstruction and model refinement

Supplementary information

Supplementary Figures

This file contains Supplementary Figure 1. (PDF 140 kb)

Ligand-induced conformational transitions of GlyR

This video shows a morph of the GlyR from the strychnine-bound to the glycine/ivermectin-bound state, via the glycine-bound state. Shown on the left side of the video is the view from the cytoplasmic side of the membrane and on the right side is the view parallel to the membrane. One subunit is highlighted. (AVI 11206 kb)

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Du, J., Lü, W., Wu, S.et al. Glycine receptor mechanism elucidated by electron cryo-microscopy.Nature526, 224–229 (2015). https://doi.org/10.1038/nature14853

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Editorial Summary

Glycine receptor mechanism

Eric Gouaux and colleagues have determined the high-resolution electron cryo-microscopy structure of strychnine-sensitive glycine receptor (GlyR) from zebrafish, bound to agonists or antagonists to reveal the conformational changes that take place when the channel opens. GlyRs mediate neurotransmission throughout the spinal cord and brainstem and their dysfunction is linked to multiple neurological disorders, including autism and hyperekplexia. Also in this issue ofNature, Xin Huanget al. report the X-ray crystal structure of the human GlyR in the presence of the antagonist strychnine.

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