Structural basis for σ1 receptor ligand recognition

Abstract

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.

Fig. 1. Crystal structures of human σ₁ receptor bound to the classical antagonists haloperidol and NE-100.

| a, σ₁ ligand pharmacophore, based on the work of Glennon et al. Representative σ₁ ligands are shown below. b, The overall structure of the human σ₁ receptor (PDB 5HK1). c, The structure of a single σ₁ monomer, with the secondary structural elements labeled. d and e, The binding pocket of the human σ₁ receptor (blue) binding in complex with haloperidol (orange, PDB ID: 6DJZ) (d) and NE-100 (light green, PDB ID: 6DK0) (e).

Fig. 2. | Crystal structure of the human σ₁ receptor bound to the classical agonist (+)-pentazocine.

In all panels, the structure of human σ₁ receptor bound to (+)-pentazocine (PDB ID: 6DK1) is shown in orange, and the structure of the human σ₁ receptor bound to PD 144418 (PDB ID: 5HK1) is show in blue. The ligands (+)-pentazocine and PD 144418 are shown in yellow and cyan, respectively. Wire mesh represents Fo-Fo density, where green mesh corresponds to regions where there is more electron density in the (+)-pentazocine-bound structure relative to the PD 144418-bound structure, and red mesh is the opposite. a, An alignment of the overall structures of σ₁ receptor bound to PD 144418 and (+)-pentazocine, with helix α4 highlighted by a red box. b, A close up of helix α4 alignment that was highlighted in a, shown in a stick representation with Fo-Fo density contoured at 2.5σ. c, An alignment of the (+)-pentazocine and PD 144418-bound structures in the binding pocket. d. Helix α4 position in simulation for unliganded (grey), (+)-pentazocine-bound (orange), and haloperidol-bound (blue) conditions. Multiple simulation frames comprising approximately 1 μs of simulation per condition are shown.

Fig. 3. | kinetic analysis of ligand binding to the σ₁ receptor

a, Association of [³H](+)-pentazocine with the σ₁ receptor measured in SPA format at 23 °C. The six ligand concentrations assayed are 300 nM (red), 100 nM (orange), 30 nM (yellow), 10 nM (green), 3 nM (blue), and 1 nM (violet). The best-fit monophasic curve for each concentration is shown as a dotted black line, and the best-fit biphasic curve for each concentration is shown as solid black lines. Error bars represent SEM. Data shown are representative from three independent experiments performed in duplicate. b and c, Residual plots for the monophasic (b) and biphasic (c) curves and the individual data points shown in a. Colors differentiate concentration of [³H](+)-pentazocine used, and are the same as in a. d, Dissociation of [³H](+)-pentazocine from the σ₁ receptor in SPA format at 23 °C. Colors represent initial [³H](+)-pentazocine concentrations as denoted in a. Solid black lines represent the best-fit monophasic exponential decay curve. Error bars represent SEM. Data shown are representative of two independent experiments performed in duplicate.

Fig. 4. | Molecular dynamics simulation reveals a putative binding pathway for (+)-pentazocine

a. Binding pathway of (+)-pentazocine, with the “lid” region shown in red and the beta strands that separate shown in purple. From left to right: simulation begins with an unliganded receptor, where the “lid” region then opens. Next, the interior of the receptor opens, and the ligand enters and binds through this opening. b. A simulation frame after the ligand has entered the binding pocket (tan), compared to a frame initiated from the crystal structure with the ligand bound (blue). Protein backbone is shown for the binding simulation in grey. Helix α4 is located at the top of the rendering.