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Structural principles of distinct assemblies of the human α4β2 nicotinic receptor

Naturevolume 557pages261265 (2018) | Download Citation


Fast chemical communication in the nervous system is mediated by neurotransmitter-gated ion channels. The prototypical member of this class of cell surface receptors is the cation-selective nicotinic acetylcholine receptor. As with most ligand-gated ion channels, nicotinic receptors assemble as oligomers of subunits, usually as hetero-oligomers and often with variable stoichiometries1. This intrinsic heterogeneity in protein composition provides fine tunability in channel properties, which is essential to brain function, but frustrates structural and biophysical characterization. The α4β2 subtype of the nicotinic acetylcholine receptor is the most abundant isoform in the human brain and is the principal target in nicotine addiction. This pentameric ligand-gated ion channel assembles in two stoichiometries of α- and β-subunits (2α:3β and 3α:2β). Both assemblies are functional and have distinct biophysical properties, and an imbalance in the ratio of assemblies is linked to both nicotine addiction2,3 and congenital epilepsy4,5. Here we leverage cryo-electron microscopy to obtain structures of both receptor assemblies from a single sample. Antibody fragments specific to β2 were used to ‘break’ symmetry during particle alignment and to obtain high-resolution reconstructions of receptors of both stoichiometries in complex with nicotine. The results reveal principles of subunit assembly and the structural basis of the distinctive biophysical and pharmacological properties of the two different stoichiometries of this receptor.

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We thank D. Cawley at OHSU for production of monoclonal antibodies, X. Bai for EM discussion, Y. Jiang for the use of the oocyte rig, C. Noviello for assistance in EM data collection and all members of the Hibbs Laboratory and M. Horvath for discussion. Cryo-EM data were collected at the UT Southwestern Medical Center Cryo-Electron Microscopy Facility, which is funded in part by CPRIT Core Facility Support Award RP170644. We thank D. Nicastro and Z. Chen for support in facility access and data acquisition and W. Chiu for cryo-EM training and resources in the National Center for Macromolecular Imaging (NCMI) at Baylor College of Medicine. NCMI is supported by NIH Grants P41GM103832 and R01GM079429. R.M.W. acknowledges support from the Sara and Frank McKnight Fund for Biochemical Research and the NIH (T32GM008203). R.E.H. is supported by a McKnight Scholar Award, The Welch Foundation (I-1812) and the NIH (DA037492, DA042072, and NS095899).

Reviewer information

Nature thanks R. Aricescu, R. Dutzler and M. Jansen for their contribution to the peer review of this work.

Author information

Author notes

  1. These authors contributed equally: Richard M. Walsh Jr, Soung-Hun Roh.


  1. Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX, USA

    • Richard M. Walsh Jr
    • , Anant Gharpure
    • , Claudio L. Morales-Perez
    • , Jinfeng Teng
    •  & Ryan E. Hibbs
  2. Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX, USA

    • Richard M. Walsh Jr
    • , Anant Gharpure
    • , Claudio L. Morales-Perez
    • , Jinfeng Teng
    •  & Ryan E. Hibbs
  3. Department of Bioengineering and BioX Program, Stanford University, Stanford, CA, USA

    • Soung-Hun Roh
  4. Biosciences Division, SLAC National Accelerator Laboratory, Menlo Park, CA, USA

    • Soung-Hun Roh


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R.M.W. expressed and purified the protein, made the EM samples, collected the EM data, processed the EM data with assistance from S.-H.R., built and refined the models and performed the binding assays. A.G. processed an initial EM dataset revealing the presence of two stoichiometries. C.L.M.-P. performed the mAb and Fab characterization. J.T. performed the electrophysiology. R.M.W. and R.E.H. wrote the manuscript with assistance from S.-H.R.; R.E.H. oversaw all aspects of the project. All authors gave feedback on the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Ryan E. Hibbs.

Extended data figures and tables

  1. Extended Data Fig. 1 Biochemistry, binding, electrophysiology and α4β2–Fab interactions.

    a, Size-exclusion chromatogram of α4β2–Fab complex and SDS–PAGE analysis of complex purification (V0, void volume). b, Saturation binding experiments with [3H]-nicotine and a mixture of receptor subunit stoichiometries. Grey squares denote samples with receptor plus Fab and blue circles denote samples with receptor alone. Receptor alone Kd = 7.7 nM (95% confidence interval (CI), 6.9–8.6 nM) and receptor plus Fab Kd = 17.0 nM (95% CI, 10.2–26.6 nM). Receptor alone and receptor plus Fab both exhibited a Hill slope of approximately 1 (1.14 and 1.09, respectively). Plotted results are from a representative experiment performed in triplicate. c, Representative two-electrode voltage clamp (−60 mV) recordings of oocytes injected with cRNAs for the α4 and β2 subunits in ratios to bias assembly,1α:5β (top two traces) and 5α:1β (bottom two traces) for the 2α:3β and 3α:2β assemblies, respectively. The experiments were performed in the presence and absence of Fab to assess the effect of Fab on receptor gating. Each oocyte was perfused with 1 µM nicotine solution for about 2 min, washed with bath solution for 10 min and then perfused for about 2 min with 1 µM nicotine ± Fab. For samples where Fab was included, 1 μM final [Fab] was added directly to the oocyte bath following the first perfusion and allowed to incubate for 1.5 min before perfusing with a second solution containing 1 µM nicotine + 0.1 μM Fab. d, Bar graph quantifying peak currents before and after adding Fab. Currents were normalized to the amplitude of the first nicotine application. Appreciable current rundown was observed (assessed by applying nicotine without Fab as the second application). Change in peak current is similar in the presence and absence of Fab for both stoichiometries, suggesting no substantial effect on gating. n, number of oocytes; error bars, s.d. from mean. eh, Fab–β-subunit interactions at the α–β, β–β, α–β, and α–α interfaces, respectively. With one exception, the Fab molecules interact exclusively with a single β-subunit. The exception is at the β–β interface, where the Fab on the complementary β-subunit forms one potential interaction with the preceding β-subunit. This interaction is displayed as a dashed line (f, inset). The conformation of the principal loop C at the β–β interface is indistinguishable from those where Fab is not interacting, suggesting that Fab does not affect the loop C conformation. Subunits are coloured as in Fig. 1

  2. Extended Data Fig. 2 Cryo-EM image processing procedure.

    a, Representative micrograph of the α4β2–Fab complex (scale bar, 50 nm). Boxed regions on the right are magnified to show representative particle images (top/bottom and side views). b, Images of selected 2D classes from reference-free 2D classification by RELION. White arrowheads, Fab fragments. c, Overview of the image processing procedure (see Methods).

  3. Extended Data Fig. 3 Three-dimensional reconstructions of the 2α:3β and 3α:2β assemblies.

    a, Angular distribution histogram of 2α:3β assembly projections. b, FSC of 2α:3β assembly maps before (black) and after (blue and red) masking. Two soft masks were used: one (red) mask that included only the receptor and one (blue) mask that included the whole 2α:3β–Fab complex. When a mask was used, the FSC curve was corrected for masking effects during the RELION post-processing procedure. Masks used in FSC calculations are shown in c for the whole 2α:3β–Fab complex (top) and receptor only (bottom), superimposed on the map in question. d, Local resolution of the 2α3β–Fab reconstruction estimated by ResMap33. Shown is the combined map, which has not been sharpened or filtered. eh, As in ad but for the 3α:2β assembly.

  4. Extended Data Fig. 4 3α:2β assembly model-map validation.

    a, b, FSC curves for cross-validation between the maps and the models with (a) and without Fab fragments (b) for the 3α:2β assembly. Curves for final model versus summed map (full) in black, for model versus half map (work) in red, and for model versus half map not used for refinement (free) in blue. For validation of receptor alone (without Fab), maps were segmented to exclude the Fab fragments. c, EM density segments of the 3α:2β assembly for a representative α4 subunit. Density map and model for α4 subunit (left). Representative density for extracellular and transmembrane spanning regions (right). Regions are numbered and helices are labelled. Maps were sharpened with a single B factor at −150 Å2. d, As in c, but for a representative β2 subunit.

  5. Extended Data Fig. 5 2α:3β assembly model–map validation.

    a, b, FSC curves for cross-validation between the maps and the models with (a) and without Fab fragments (b) for the 2α:3β assembly. Curves for final model versus summed map (full) in black, for model versus half map (work) in red, and for model versus half map not used for refinement (free) in blue. For validation of receptor alone (without Fab) maps segmented to exclude the Fab fragments were used. c, EM density segments of the 2α:3β assembly for a representative α4 subunit. Density map and model for α4 subunit (left). Representative density for extracellular and transmembrane spanning regions (right). Regions are numbered and helices are labelled. Maps were sharpened with a single B factor at −170 Å2. d, As in c, but for a representative β2 subunit.

  6. Extended Data Fig. 6 Comparison of map and model of 2α3β by X-ray crystallography at 3.9 Å resolution and by cryo-EM at 3.4 Å resolution, and comparison of subunit backbone conformations.

    a, The X-ray electron density map (left) and cryo-EM map (right) are displayed in a linear representation (unwrapped) to illustrate the overall resolvability of all five subunits: two α4 subunits (green) and three β2 subunits (blue). Red circles indicate the locations of crystal contacts on the X-ray electron density map (left) or Fab binding sites on cryo-EM density map (right). b, X-ray structure of the 2α:3β assembly (PDB accession 5KXI8) after alignment to the cryo-EM structure (this study) shown in ribbon representation and coloured according to Cα r.m.s.d. values (from blue (low) to red (high)). While the areas involved in crystal contacts displayed relatively higher r.m.s.d. values for Cα ( > 2 Å), the Fab binding location shows r.m.s.d. values for Cα of < 1 Å. This result implies that zones of protein–protein contacts in the crystal lattice may substantially affect local structure on the pentamer, but Fab binding in the cryo-EM study did not result in substantial structural change, consistent with the binding assays and electrophysiological results (Extended Data Fig. 1). c, Fit of X-ray model (5KXI) to X-ray electron density map (left) and cryo-EM model to cryo-EM density map (middle) shown as selected areas for high r.m.s.d. (loop C) and low r.m.s.d. (M2 helix), respectively. Right column shows superposition of X-ray and cryo-EM model at the selected areas. dh, Subunit superpositions. d, Comparison of all α4 subunits in the 2α:3β and 3α:2β assemblies reveals no substantial conformational differences in α-carbon backbones. e, Superposition of all β2 subunits in the two assemblies reveals a domain motion of the TMD relative to the ECD of the subunits that comprise the β–β interface. The rotation of 4.4° between the two β-subunits that compose the β–β interface is denoted by black double-headed arrows. The principal β-subunit (β2 + ) is coloured magenta while the complementary β-subunit (β2 −) is shown in cyan. fh, Superposition of α4 (green) with the three distinct β2 conformations: principal β–β (magenta) complementary β–β (cyan) and α–β–α (grey). The conformation of the principal β–β (magenta) subunit is distinct from every other subunit in the 2α:3β and 3α:2β assemblies. The conformation of the complementary (−) subunit of the β–β interface is rotated towards the pore axis, accommodating the marked conformational change of the principal β2 subunit away from the pore axis. This conformational change results in the complementary β–β (cyan) subunit adopting a backbone conformation similar to α4 subunits.

  7. Extended Data Fig. 7 Fenestration at β–β interface.

    Extracellular fenestration unique to this class of interface. Inset of fenestration with distances indicated by dashed lines and side chains surrounding fenestration shown as sticks. Two conserved glutamic acid residues in close proximity to this gap at the β–β interface (yellow) have been implicated in Ca2+ potentiation of nicotinic receptors49.

  8. Extended Data Fig. 8 Basis of α4β2 heteromeric assembly.

    a, Cartoon representation of top view of observed (2α:3β and 3α:2β) and computational (4α:1β, 1α:4β, 5α and 5β) α4β2 pentameric assemblies. Assemblies on the top row (3α:2β, 4α:1β, and 5α) are arranged by increasing α4 composition. Assemblies on the bottom row (2α:3β, 1α:4β and 5β) are arranged by increasing β2 composition. Agonist binding sites are denoted by red circles. Buried interface areas (Å2) for the interfaces analysed in b, c are listed below each pentameric assembly. Subunits are coloured in a as described below for b, c. b, Superposition of α–α from 3α:2β and final α–α interface in 5α homopentamer. Principal subunits (grey) were superimposed to highlight differences at the interface. Complementary (−) subunits are coloured light green for α–α from 3α:2β and dark green for the α–α interface in the 5α homopentamer. Sticks are displayed for amino acid clashes that have greater than 1.5 Å overlap assessed by Molprobity. c, Superposition of the β–β interface from 2α:3β and final β–β interface in the 5β homopentamer. Principal subunits (grey) were superimposed to highlight differences at the interface. Complementary (−) subunits are coloured light blue for the β–β interface from the 2α:3β assembly and dark blue for the final β–β interface in the 5β homopentamer.

  9. Extended Data Fig. 9 Ligand binding site comparisons.

    a, Stereo images of nicotine bound at the α–α interface. Subunits are coloured as in Fig. 1. Density (purple mesh) is displayed at a threshold of 0.0306 in Chimera. Electrostatic interactions denoted as dashed magenta lines. Binding pocket residues and nicotine displayed as sticks. b, Ligplot of nicotine bound at the α–α and α–β interfaces (left and right, respectively). Residue contacts within 4.5 Å are displayed in red, residue displayed in purple (His116) is within 5.5 Å. c, Stereo images of nicotine bound at the α–β interface. Density (purple mesh) is displayed at a threshold of 0.0306 in Chimera. Hydrogen bonds denoted as dashed magenta lines. Binding pocket residues and nicotine displayed as sticks. d, e, Stereo image overlays of nicotine bound at the α–α (magenta) and α–β interfaces (cyan). Ligand density is displayed as transparent surfaces. Density is displayed at a threshold of 0.406 for the α–α (magenta) and 0.500 for the α–β (cyan) interface in Chimera. e, Ribbon removed for clarity, rotated 180° relative to d. Key residues implicated in the difference in sensitivity between the classical α–β neurotransmitter binding site (high sensitivity V111/F119/L121) and the unique α–α binding site (low sensitivity H116/Q124/T126) are labelled.

  10. Extended Data Fig. 10 Comparison of ions located in the pore above the constriction point.

    a, Top view of EM density map of ion in the pore of the 3α:2β assembly. Density (blue mesh) is displayed at a threshold of 0.027 in Chimera. Subunits are coloured as in Fig. 1. Modelled Na+ ion is represented as a purple sphere. Nearest residues on the M2 α-helices are indicated. b, As a, but for the 2α:3β assembly. c, Side view of EM density map of ion in the pore of the 3α:2β assembly. Two subunits (one α and one β) removed for clarity. Colours and residues indicated as in a. d, As c, but for the 2α:3β assembly. e, Superposition of constriction region of 2α:3β (light blue) and 3α:2β (light green) assemblies.

  11. Extended Data Fig. 11 Comparison of putative cholesterol binding orientations.

    a, b, EM density map showing cholesterol sites from all TMD interfaces in the 3α:2β assembly and 2α:3β assembly, respectively. Pentagons (top) are coloured to illustrate subunits composing the displayed interface. c, Stereo image of representative cholesterol binding site with α-subunit on principal side ( + ). When the principal side ( + ) is an α4 subunit, both molecules tilt towards the principal face. One residue from the complementary side β2 (−), Y232, contacts both cholesterol molecules at the α–β interface (at the α–α interface the equivalent residue Y240 in the complementary (−) α4 contacts both cholesterol molecules at the interface; not shown). d, Stereo image of representative cholesterol binding site with β-subunit on principal side ( + ). When the principal side ( + ) is a β2 subunit, both cholesterol molecules are oriented orthogonal to the plane of the membrane. One residue from the principal ( + ) β2 subunit, V288, makes contacts with both cholesterol molecules at the β–α and β–β (not shown) interfaces. Cholesterol and interacting side chains are shown as sticks. Density maps in panels ad are displayed at a threshold of 0.025 in Chimera. e, Alignment of nicotinic receptor subunits for region encompassing putative cholesterol sites. Residues in cholesterol binding pocket are boxed and amino acid position implicated in differential cholesterol binding is highlighted in magenta. Uniprot accession IDs are provided40. Residues mapped using a photoreactive cholesterol analogue in Torpedo californica (TC P02710)26,27 are highlighted in cyan. All other sequences are from Homo sapiens (HS P43681, P11230, P17787, Q05901, P30926 for α4 and β1–4, respectively). f, Superposition of β–α (yellow) from α4β2 and α1–α1 (blue) GABAA–GLIC chimaera (PDB accession 5OSC25) TMD interface to compare putative binding sites. Pregnenolone sulfate, CHS and cholesterol are shown as sticks.

  12. Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics

Supplementary information

  1. Supplementary Information

    The uncropped gel shown in Extended Data Fig. 1

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