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Crystal structure of human glycine receptor-α3 bound to antagonist strychnine


Neurotransmitter-gated ion channels of the Cys-loop receptor family are essential mediators of fast neurotransmission throughout the nervous system and are implicated in many neurological disorders. Available X-ray structures of prokaryotic and eukaryotic Cys-loop receptors provide tremendous insights into the binding of agonists, the subsequent opening of the ion channel, and the mechanism of channel activation1,2,3,4,5,6,7,8. Yet the mechanism of inactivation by antagonists remains unknown. Here we present a 3.0 Å X-ray structure of the human glycine receptor-α3 homopentamer in complex with a high affinity, high-specificity antagonist, strychnine. Our structure allows us to explore in detail the molecular recognition of antagonists. Comparisons with previous structures reveal a mechanism for antagonist-induced inactivation of Cys-loop receptors, involving an expansion of the orthosteric binding site in the extracellular domain that is coupled to closure of the ion pore in the transmembrane domain.

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Figure 1: Architecture of the GlyRα3 bound to strychnine.
Figure 2: Orthosteric binding site occupied by the antagonist strychnine.
Figure 3: The ion channel of the GlyRα3–strychnine complex in a closed state.
Figure 4: Structural coupling at the ECD–TMD interface.
Figure 5: Conformational changes in pLGICs.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the GlyRα3–strychnine complex have been deposited in the Protein Data Bank (PDB) under accession number 5CFB.


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We thank G. Ranieri and R. Walter at Shamrock Structures and the staff at beamlines 08-ID at the Canadian Light Source and 22-ID at the Advanced Photon Source for data collection. We are grateful to Z. Wang and J. Gingras for reviewing the manuscript.

Author information

Authors and Affiliations



The authors have jointly contributed to project design, data analysis and manuscript preparation. P.L.S. performed initial construct design and purification experiments, structure solution, model building, and structural analysis; X.H. performed protein purifications, crystallization, model building, and structural analysis; H.C. performed cloning and expression experiments; K.M. performed SPR and ITC binding studies; S.S. performed functional testing; P.L.S. and X.H. wrote the manuscript with help from K.M., S.S., and H.C.

Corresponding authors

Correspondence to Xin Huang or Paul L. Shaffer.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Strychnine binding to GlyRα3cryst in detergent micelles.

a, Binding thermodynamics and stoichometry of strychnine to GlyRα3cryst analysed by ITC. The individual peaks from titrations are integrated and presented in a Wiseman plot. An appropriate binding model is chosen and the isotherm is then fitted to yield the binding enthalpy ΔH, the dissociation constant Kd, and the stoichiometry n. b, Binding kinetics of strychnine to GlyRα3cryst measured by SPR spectroscopy.

Extended Data Figure 2 Glycine dose–response to GlyRα3cryst in HEK293T cells.

Glycine dose–response curve for BacMam baculovirus-transduced HEK293T cells expressing either wild-type human GlyRα3 (GlyRα3, filled circles) or GlyRα3cryst (open circles) measured by membrane potential dye assay (see Methods for details). Each data point represents a value of n = 4–6 and normalized to a maximum glycine response observed at 2 mM glycine concentration. Glycine EC50 for GlyRα3 was calculated at 150 ± 10.6 µM (n = 4, 95% confidence interval) and GlyRα3cryst at 16.4 ± 1.2 µM (n = 5, 95% confidence interval).

Extended Data Figure 3 Architecture of the GlyRα3 bound to strychnine and crystallographic packing of GlyRα3cryst.

a, The GlyRα3–strychnine complex viewed from the extracellular side of the membrane down the pore axis, perpendicular to the membrane. Strychnine is bound at the interface between subunits. b, The GlyRα3–strychnine complex viewed from the intracellular side down the pore axis. The M2 helices are shown lining the pore. c, d, Packing of the GlyRα3–strychnine complex. Receptor in the asymmetric unit is coloured by subunit, with subunit A in pale green and subunit B in cyan. Strychnine molecules are shown as spheres and are coloured to match their associated principal subunit. Symmetry-related receptors are coloured grey. The interface between subunits A and B is completely exposed to solvent in the crystal lattice, and this interface was used in the making of all figures for this paper to avoid any potential crystal-packing artefacts.

Extended Data Figure 4 Strychnine binding to GlyRα3.

a, b, Two views of the strychnine-binding pocket showing 2FoFc omit electron density maps. The principal subunit is coloured pale green, the complementary subunit is cyan, and strychnine is grey. Strychnine was omitted from map calculations. Map is contoured at 1.0σ. The hydrogen bond between the backbone carbonyl of Phe159 and the tertiary nitrogen of strychnine is shown as a dotted line. c, d, Same views and representations as in a and b, but FoFc omit electron density is shown. Contour level of map is 3.0σ. e, Chemical structure of strychnine. Ring numbering is denoted by red Roman numerals. Carbon and nitrogen historic numbering conventions are denoted by blue numbers. f, Solvent-accessible surface area of the complementary side of the binding interface coloured by electrostatic potential. The complementary subunit interface has a positive electrostatic potential to interact with the partial negative charge of the lactam oxygen of strychnine.

Extended Data Figure 5 Sequence alignment of GlyRα3cryst with representative eukaryotic Cys-loop receptor family members.

Residue conservation is indicated by grey and black highlights. Site of N-linked glycosylation of GlyRα3cryst is indicated by the orange hexagon. Residues involved in binding of strychnine are indicated by green (the principal subunit) and cyan (the complementary subunit) dots. Red triangles above residues indicate mutations in GlyRα1 or GlyRβ that cause hyperekplexia. Signal peptides have been removed from all protein sequences. Secondary structure elements are denoted by cylinders (helices) and arrows (strands) above the alignment. The alignment was generated using ClustalW. Protein sequences are from the following entires: human GlyRα1 (Uniprot P23415), human GlyRβ (Uniprot 48167), GluClcryst (PDB accession number 4TNV), GABAAR-β3cryst (PDB accession number 4COF), and 5HT3A (PDB accession number 4PIR).

Extended Data Figure 6 Structural alignment of pLGICs.

a, Structures of GlyRα3cryst (green), apo-GluClα (slate), and glutamate- and ivermectin-bound GluCLα (violet) aligned using their ECDs. b, Close-up view of the loop C, rotated 45 ° clockwise along the horizontal axis (left to right) to optimize viewing. c, Close-up view of loops β6–β7 and β1–β2. d, Table showing regions of GlyRα3 used for structural alignments, as well as resulting number of overlapped Cα atoms and root mean squared deviation distances. PDB accession numbers are listed for each reference structure.

Extended Data Figure 7 Representative electron density for the ECD–TMD interface.

Final 2FoFc electron density map around the region of the ECD–TMD interface is shown, contoured at 1.0σ. Protomer A is shown in pale green, protomer B in cyan, and protomer C in magenta. Important secondary structure elements and loops are noted.

Extended Data Table 1 Crystallographic and structure refinement statistics

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Huang, X., Chen, H., Michelsen, K. et al. Crystal structure of human glycine receptor-α3 bound to antagonist strychnine. Nature 526, 277–280 (2015).

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