Crystal structure of the α1B-adrenergic receptor reveals molecular determinants of selective ligand recognition

Abstract

α-adrenergic receptors (αARs) are G protein-coupled receptors that regulate vital functions of the cardiovascular and nervous systems. The therapeutic potential of αARs, however, is largely unexploited and hampered by the scarcity of subtype-selective ligands. Moreover, several aminergic drugs either show off-target binding to αARs or fail to interact with the desired subtype. Here, we report the crystal structure of human α_1BAR bound to the inverse agonist (+)-cyclazosin, enabled by the fusion to a DARPin crystallization chaperone. The α_1BAR structure allows the identification of two unique secondary binding pockets. By structural comparison of α_1BAR with α₂ARs, and by constructing α_1BAR-α_2CAR chimeras, we identify residues 3.29 and 6.55 as key determinants of ligand selectivity. Our findings provide a basis for discovery of α_1BAR-selective ligands and may guide the optimization of aminergic drugs to prevent off-target binding to αARs, or to elicit a selective interaction with the desired subtype.

Fig. 1. Structure of α_1BAR_XTAL bound to (+)-cyclazosin and overview of the ligand-binding site.

a Chemical structure of (−)- and (+)-cyclazosin. N1 is expected to be mostly protonated at the crystallization pH of 6.0 (see “Methods”) as well as at physiological pH, and the resulting positive charge will be delocalized over the quinazoline ring system^–. b Structure of α_1BAR_XTAL bound to (+)-cyclazosin. For clarity, DARPin D12 has been omitted. (+)-Cyclazosin is depicted as van der Waals spheres. The two orientations observed for the furan-2-yl-methanone substituent of (+)-cyclazosin are colored in cyan and pale cyan, respectively, and are indicated by a black curved arrow. Oxygen, nitrogen, and sulfur atoms are depicted in red, blue, and yellow, respectively. ECL, extracellular loop; ICL, intracellular loop. c Surface representation of the (+)-cyclazosin binding site in α_1BAR_XTAL. ECL1–3 are shown as surface and as cartoon; (+)-cyclazosin is shown as sticks. The orthosteric binding site (OBS) has been approximated on the basis of the β₂AR-epinephrine complex (see panel d and main text). d Comparison of the binding modes of (+)-cyclazosin in α_1BAR_XTAL and epinephrine in β₂AR (PDB ID: 4LDO).

Fig. 2. (+)-Cyclazosin binding pocket in α_1BAR_XTAL.

a Detailed view of the (+)-cyclazosin binding site. (+)-Cyclazosin is shown as sticks in cyan, with the two alternative orientations observed for the furan-2-yl-methanone substituent (highlighted by a dashed red ellipse) colored in cyan (on the left) and pale cyan (on the right), respectively. Receptor residues are shown as sticks in pale green except for the F334→L mutation, which is colored in dark gray and is indicated by an asterisk. V197 is shown to Cβ only because its side chain is not resolved in the electron density map. Hydrogen bonds are depicted as dashed blue lines. Oxygen, nitrogen, and sulfur atoms are depicted in red, blue, and yellow, respectively. b MD simulation of α_1BAR_XTAL-MD-V333-F334. The plots on the right indicate the structural stability of (+)-cyclazosin and F334_MD throughout the simulation. RMSD, root-mean-square deviation; g⁻, gauche minus conformation of the χ₁ dihedral angle. For (+)-cyclazosin, RMSD values were calculated on all atoms. A representative snapshot of the final nanosecond of the simulation is depicted on the left, viewed from the same perspective as in panel a. (+)-Cyclazosin is colored in teal; F334_MD is colored in dark green. c Schematic representation of the (+)-cyclazosin binding site. OBS, orthosteric binding site; SBPs, secondary binding pockets. A black curved arrow indicates the two orientations observed for the furan-2-yl-methanone moiety. Note that residues C195^45.50, G196^45.51, and V197^45.52 belong to ECL2, which forms crystal contacts. Source data are provided as a Source Data file.

Fig. 3. Comparison of the ligand-binding pockets of α_1BAR_XTAL bound to (+)-cyclazosin and α_2CAR-RS79948.

a Alignment of residues delineating the binding pockets of (+)-cyclazosin in α_1BAR_XTAL and of RS79948 in α_2CAR (PDB ID: 6KUW). Non-conserved residues between human α₁ARs and α₂ARs are highlighted by solid blue rectangles, whereas dashed blue rectangles highlight partially non-conserved residues. Underlined black residues interact with the cognate ligand, whereas non-underlined black residues do not, but are nonetheless within 5 Å of it (ligand-receptor interactions are listed in Supplementary Tables 4 and 5). Gray residues are >5 Å away from the cognate ligand. Aromatic residues are highlighted in orange, hydrophobic residues in yellow, polar residues in green, Cys in yellow-green, acidic residues in red, basic residues in blue. b Superposition of α_1BAR_XTAL bound to (+)-cyclazosin with α_2CAR-RS79948, focusing on the non-conserved residues within the ligand-binding pocket (cf. panel a). Receptor residues are shown as sticks; ligands are shown in ball-and-stick representation. V197 is shown to Cβ only because its side chain is not resolved in the electron density map. A black curved arrow indicates the two orientations observed for the furan-2-yl-methanone substituent of (+)-cyclazosin. Oxygen, nitrogen, and sulfur atoms are depicted in red, blue, and yellow, respectively.

Fig. 4. Molecular determinants and structural basis for the selective binding of RS79948 to α_2CAR over α_1BAR.

a Affinity of RS79948 for α_1BAR, α_2CAR, and chimeric α_1BAR-α_2C mutants. Single amino acid substitutions in α_1BAR are indicated in the construct names and correspond to the α_2CAR sequence at either one of positions 3.28, 3.29, 45.52, or 6.55. The α_1BAR-α_2C(YLLY) chimera corresponds to the quadruple mutant. Data are shown as mean values ± standard error of the mean (SEM) of 3–6 independent experiments performed in triplicate. The underlying data points are depicted as black diamonds, and the exact n, SEM, and 95% confidence interval of the mean are reported in Supplementary Table 6. Differences in affinities were evaluated by a statistical test as detailed in Supplementary Table 7. b Structural role of Y127^3.28, L128^3.29, L204^45.52, and Y402^6.55 in the binding of RS79948 to α_2CAR (PDB ID: 6KUW). TM1, ECL3, and TM7 have been omitted for clarity. Receptor residues are shown as van der Waals spheres and as sticks, except for D131^3.32, which is shown as sticks only; RS79948 is shown as sticks. Oxygen, nitrogen, and sulfur atoms are depicted in red, blue, and yellow, respectively. c Superposition of α_1BAR_XTAL bound to (+)-cyclazosin and α_2CAR-RS79948 (PDB ID: 6KUW), focusing on the residues outlined in panel b. (+)-Cyclazosin, TM1, TM2, ECL1, ECL3, and TM7, have been omitted for clarity. Receptor residues and RS79948 are shown as sticks. V197 is shown to Cβ only because its side chain is not resolved in the electron density map. Source data are provided as a Source Data file.

Fig. 5. Molecular determinants for the preferred binding of piperazinyl quinazolines to α₁ARs over α₂ARs.

a–c Affinity of (a) QAPB, (b) prazosin, and (c) cyclazosin for α_1BAR, α_2CAR, and chimeric α_1BAR-α_2C mutants. Single amino acid substitutions in α_1BAR are indicated in the construct names and correspond to the α_2CAR sequence at either one of positions 3.28, 3.29, 45.52, or 6.55. The α_1BAR-α_2C(YLLY) chimera corresponds to the quadruple mutant. All three ligands share a common piperazinyl 4-amino-6,7-dimethoxyquinazoline scaffold, which is highlighted in red in their chemical structures. Data are shown as mean values ± SEM of 3–6 independent experiments performed in triplicate. The underlying data points are depicted as black diamonds, and the exact n, SEM, and 95% confidence interval of the mean are reported in Supplementary Table 6. Differences in affinities were evaluated by a statistical test as detailed in Supplementary Table 7. d Superposition of α_1BAR_XTAL bound to (+)-cyclazosin and α_2CAR-RS79948 (PDB ID: 6KUW), focusing on the potential role of ECL2 in selective ligand binding. The ECL2 residues in α_2CAR that form a lid on the ligand-binding site are shown as red van der Waals spheres. (+)-Cyclazosin is depicted as sticks and as transparent van der Waals spheres, with the two orientations observed for its furan-2-yl-methanone substituent colored in cyan and pale cyan, respectively. Black arrows indicate potential hindrance exerted by ECL2 with respect to (+)-cyclazosin. Source data are provided as a Source Data file.