The σ1 receptor is a poorly understood membrane protein expressed throughout the human body. Ligands targeting the σ1 receptor are in clinical trials for treatment of Alzheimer’s disease, ischemic stroke, and neuropathic pain. However, relatively little is known regarding the σ1 receptor’s molecular function. Here, we present crystal structures of human σ1 receptor bound to the antagonists haloperidol and NE-100, and the agonist (+)-pentazocine, at crystallographic resolutions of 3.1 Å, 2.9 Å, and 3.1 Å, respectively. These structures reveal a unique binding pose for the agonist. The structures and accompanying molecular dynamics (MD) simulations identify agonist-induced structural rearrangements in the receptor. Additionally, we show that ligand binding to σ1 is a multistep process that is rate limited by receptor conformational change. We used MD simulations to reconstruct a ligand binding pathway involving two major conformational changes. These data provide a framework for understanding the molecular basis for σ1 agonism.
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Atomic coordinates and crystallographic structure factors have been deposited in the Protein Data Bank under accession codes PDB 6DJZ (σ1 receptor–haloperidol complex), PDB 6DK0 (σ1 receptor–NE-100 complex), and PDB 6DK1 (σ1 receptor–(+)-pentazocine complex). All other data that support the findings of this study are available from the corresponding author upon reasonable request.
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We thank B. Kelly for performing preliminary simulations of the σ1 receptor trimer and Advanced Photon Source GM/CA beamline staff for excellent technical support in crystallographic data collection. We also thank F. Kim (Drexel University) for generously providing (+)-pentazocine for crystallographic studies. This work was supported by a Klingenstein-Simons Fellowship in Neuroscience (A.C.K.), National Institutes of Health grants 1R01GM119185 (A.C.K.) and 1R01GM127359 (R.O.D.), the Winthrop Fund/Harvard Brain Science Initiative (A.C.K.), and National Science Foundation Graduate Research Fellowship awards DGE1745303 (H.R.S.) and DGE1656518 (R.M.B.).
The authors declare no competing interests.
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Integrated supplementary information
Supplementary Figure 1 The structure of antagonist-bound σ1 receptor is consistent among four crystal structures.
a, The amino acid sequence of human σ1 receptor annotated by secondary structure, based on the available crystal structures. b-e, A view of the σ1 receptor ligand binding pocket, with the protein shown in grey. All co-crystallized ligands are shown as sticks and superimposed on one another. They include PD 144418 (cyan), 4-IBP (salmon), haloperidol (orange), and NE-100 (light green)
Supplementary Figure 2 Representative electron density for the crystal structures of σ1 receptor bound to haloperidol, NE-100, and ( + )-pentazocine.
a,c,e, Composite omit 2Fo-Fc electron density contoured at 1.0σ for σ1 receptor bound to haloperidol (a), NE-100 (b), and ( + )-pentazocine (e), depicting the transmembrane domain from chain B, including residues 1–23. b,d,f, polder OMIT map around the ligand in chain C, for haloperidol (b), NE-100 (d), and ( + )-pentazocine (f) contoured at 2.0 σ
In all panels, the σ1 receptor backbone is shown in grey cartoon, with the α4 helix shown in orange for the ( + )-pentazocine-bound structure and blue for the PD144418-bound structure. ( + )-pentazocine and PD144418 are shown in yellow and cyan sticks, respectively. a, An overall view of the ( + )-pentazocine and PD144418-bound structures aligned with once another, with red arrows indicating the direction of the α4 helix shift seen in the ( + )-pentazocine-bound structure relative to the antagonist-bound structures. b, A closeup view of the interface between the chain C and the α4 helix of chain A, highlighting the movement of Q194/C. c, a model of (-)-pentazocine (purple) superimposed on the co-crystalized ligand ( + )-pentazocine. The two ligand were aligned by the atoms outside the benzomorphan rings to illustrate that (-)-pentazocine must adopt a different pose than ( + )-pentazocine to fit into the binding pocket. d, Glide docking results for PRE-084 (green) docked into the pentazocine-bound σ1 receptor structure (PDB ID: 6DK1)
For all plots, error bars represent SEM, unless otherwise indication. a, Dissociation curve for [3H]( + )-pentazocine from the σ1 receptor in Sf9 membranes obtained at 37 °C. Data shown are representative of more than three independent experiments performed in triplicate. b, A time course showing total (red) and nonspecific (blue) binding of 100 nM [3H]( + )-pentazocine to σ1 receptor coupled to SPA beads. Each curve is a separate vial, with total and nonspecific binding each assayed in duplicate. c, [3H]( + )-pentazocine saturation curves for σ1 receptor binding in membranes at 37 °C (blue), and in SPA format at 23 °C (red). For the membrane binding experiment, the curve is representative of two independent experiments performed in duplicate. For the SPA measurement, the shown curve is representative of three independent experiments performed in duplicate. d, The observed rate constant for the slow step of ( + )-pentazocine association with the σ1 receptor, kslow, averaged from 3 independent association experiments performed in duplicate. For final fits, kslow was constrained to be shared for all concentrations in an independent experiment, but it is shown here unconstrained to illustrate the lack of change with concentration, which was confirmed by an ANOVA test. Error bars represent standard deviation. e, Residual plots for monophasic or biphasic decay curves for [3H]( + )-pentazocine dissociation from σ1 receptor in SPA format. Colors represent different initial concentrations of [3H]( + )-pentazocine as explained in Fig. 3a. f, The observed rate constant for the fast step of ( + )-pentazocine association with the σ1 receptor, kfast, plotted against the concentration of ( + )-pentazocine used. Values were determined from the plot shown in Fig. 3a, and are representative of three independent experiments. The solid line represents a simple exponential fit to the kfast values, to illustrate the nonlinear relationship between kfast and ( + )-pentazocine concentration. g, The observed rate constant for dissociation of [3H]( + )-pentazocine from σ1 receptor, koff, with respect to the starting concentration of ( + )-pentazocine. Values for koff were determined from the plot in Fig. 3d, and are representative of two independent experiments. h, Hill plot for σ1 receptor binding to [3H]( + )-pentazocine, generated from the saturation curve shown in b. The Hill coefficient, nH, was calculated to be 0.91 and was not significantly different than 1.0
Supplementary Figure 5 SPA kinetic analysis of [3H]haloperidol binding and association of [3 H]( + )-pentazocine with σ1 receptor in membranes.
a, Association of [3H]haloperidol with the σ1 receptor in SPA format at 23 °C. Association was measured at three concentrations: 10 nM (red), 3 nM (green), and 1 nM (blue). The black dotted lines represent the best-fit monophasic exponential association curve for each concentration, while the solid lines represent the best-fit biphasic curve. Data are representative of two independent experiments performed in duplicate. b, Residual plots for the monophasic and biphasic plots shown in a, with all colors remaining the same as in a. c, Dissociation of [3H]haloperidol from the σ1 receptor in SPA format at 23 °C. Colors denote initial [3H]haloperidol concentrations as described in a. Black lines represent the best-fit monophasic exponential decay curves for each initial concentration of ligand. d, Residual plots for monophasic or biphasic decay curves for [3H]haloperidol dissociation from σ1 receptor in SPA format. Colors represent different initial concentrations of [3H]haloperidol as explained in a. e, Association curve for 1 nM (blue circles), 10 nM (green squares), or 100 nM (red triangles) [3H]( + )-pentazocine with the σ1 receptor in Sf9 membranes obtained at 37 °C. Data shown are representative of three independent experiments performed in triplicate. The solid lines represent the best fit curves for two-step association
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Schmidt, H.R., Betz, R.M., Dror, R.O. et al. Structural basis for σ1 receptor ligand recognition. Nat Struct Mol Biol 25, 981–987 (2018). https://doi.org/10.1038/s41594-018-0137-2
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