Structural characterization of binding mode of smoking cessation drugs to nicotinic acetylcholine receptors through study of ligand complexes with acetylcholine-binding protein

Abstract

Smoking cessation is an important aim in public health worldwide as tobacco smoking causes many preventable deaths. Addiction to tobacco smoking results from the binding of nicotine to nicotinic acetylcholine receptors (nAChRs) in the brain, in particular the α4β2 receptor. One way to aid smoking cessation is by the use of nicotine replacement therapies or partial nAChR agonists like cytisine or varenicline. Here we present the co-crystal structures of cytisine and varenicline in complex with Aplysia californica acetylcholine-binding protein and use these as models to investigate binding of these ligands binding to nAChRs. This analysis of the binding properties of these two partial agonists provides insight into differences with nicotine binding to nAChRs. A mutational analysis reveals that the residues conveying subtype selectivity in nAChRs reside on the binding site complementary face and include features extending beyond the first shell of contacting residues.

FIGURE 1.

A, shown are chemical structures of ligands used in this work: nicotine, cytisine, and varenicline. Bottom views of AcAChBP in complex with cytisine (B) and varenicline (C) are shown. Each chain of the pentameric assembly is colored differently. Two blown-up views of one protomer-protomer interface are provided for cytisine (left) and for varenicline (right), with interacting residues represented using sticks and placed in the δA-weighted 2mF_o − dF_c electron density map contoured at 1σ (blue mesh), carved 6.5Å around the ligands. In these blown-up views, principal face residues are in blue, whereas the complementary face residues are colored orange.

FIGURE 2.

Detailed views of the Ls- (A) or AcAChBP (B and C) binding sites are depicted. Principal face residues are colored blue, whereas complementary face residues are colored orange. Interacting residues are represented as sticks, and water molecules are shown as spheres. The nicotine-LsAChBP complex (PDB code 1UW6) (A), cytisine-AcAChBP complex (B), and varenicline-AcAChBP complex (C) are represented. D, shown is secondary structure matching superposition (53) of binding site principal faces of Ac-, Ls- (PDB code 1UW6), and BtAChBPs (PDB code 2BJ0, ligand omitted for clarity). Interesting, residues are represented as blue, silicon, and orange sticks, respectively, to outline the variability among the binding sites. For conserved residues, AcAChBP numbering is provided. E, shown is secondary structure matching superposition (53) of binding site principal faces of AcAChBP in complex with cytisine (blue) and varenicline (orange) and of LsAChBP in complex with nicotine (PDB code 1UW6). Interacting residues are represented as sticks to allow a comparison of binding modes for these ligands. For conserved residues, AcAChBP numbering is provided.

FIGURE 3.

ITC titration curves for the binding of nicotine (A), cytisine (B), and varenicline (C) to AcAChBP are provided. Nicotine binding data were recorded on a VP-ITC instrument, whereas cytisine and varenicline binding data were recorded on an ITC200 instrument. The thermodynamic profiles for nicotine, cytisine, and varenicline binding to AcAChBP (D) are displayed as a bar chart with Gibb's energy (ΔG°) in blue and the enthalpic (ΔH°) and entropic contributions (−TΔS°) in red and in green, respectively. A similar bar chart representation is provided for the comparison of nicotine binding to Ac-, Ls-, and BtAChBPs (E).

FIGURE 4.

Ribbon representation of LsAChBP in complex with nicotine (blue), cytisine (orange), varenicline (green), methyllycaconitine (gray, PDB code 2BYR) and DMXBA (pink, PDB code 2WNJ) superposed with respect to their principal face. Nicotine is represented as sticks to indicate the orientation of a full agonist in the binding site. The tip of loop C is displaced outwards by 5.7 Å when AcAChBP is bound to the α7 nAChR antagonist methyllycaconitine and by 2.7 Å when AcAChBP is bound to partial agonist DMXBA when compared with the nicotine-bound state. No significant difference in the position of loop C is measured when AcAChBP is bound to nicotine, cytisine, or varenicline.

FIGURE 5.

Structure-based sequence alignment (50) of Ac-, Ls-, and BtAChBPs with the α4, β2, and α7 nAChR subunits. Ligand binding site residues involved in nicotine binding are boxed. Principal face residues are colored and boxed in blue, whereas complementary face ligand binding residues are colored and boxed in orange. Residues that were mutated to reflect α4β2 and α7 nAChRs are indicated by a vertical green arrow.

FIGURE 6.

Bar charts representing the improvement in binding affinity of selected mutants with respect to wild type AcAChBP (A) or to AcAChBP incorporating the Y53W mutation (B). Improvement in affinity was measured as the ratio of affinities (K_D) of either wild type (A) or Y53W (B) AcAChBPs to those of other AcAChBP mutants. Errors were propagated from initial radioligand binding measurements (Table 2).