Crystal structure of Lymnaea stagnalis AChBP complexed with the potent nAChR antagonist DHβE suggests a unique mode of antagonism

Abstract

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels that belong to the Cys-loop receptor superfamily. These receptors are allosteric proteins that exist in different conformational states, including resting (closed), activated (open), and desensitized (closed) states. The acetylcholine binding protein (AChBP) is a structural homologue of the extracellular ligand-binding domain of nAChRs. In previous studies, the degree of the C-loop radial extension of AChBP has been assigned to different conformational states of nAChRs. It has been suggested that a closed C-loop is preferred for the active conformation of nAChRs in complex with agonists whereas an open C-loop reflects an antagonist-bound (closed) state. In this work, we have determined the crystal structure of AChBP from the water snail Lymnaea stagnalis (Ls) in complex with dihydro-β-erythroidine (DHβE), which is a potent competitive antagonist of nAChRs. The structure reveals that binding of DHβE to AChBP imposes closure of the C-loop as agonists, but also a shift perpendicular to previously observed C-loop movements. These observations suggest that DHβE may antagonize the receptor via a different mechanism compared to prototypical antagonists and toxins.

Figure 1. The structure of DHβE and Ls-AChBP complexed with DHβE.

(a) Structure of DHβE. (b) Cartoon diagram showing homopentameric Ls-AChBP viewed along the five-fold symmetry axis. The five subunits are shown in different colors and DHβE in red spheres representation. (c) Ligand-binding pocket at the interface of two monomers formed by the highly conserved aromatic residues Tyr89, Trp143, Tyr185, and Tyr192 from the principal side of the interface (yellow) and Trp53 from the complementary side (limon). DHβE is shown in red and an omit 2Fo-Fc map is shown at 1σ. Hydrogen bonds between DHβE and its surroundings are shown as stippled lines. A blow-up of DHßE and the omit 2Fo-Fc map shown at 1σ is provided in Fig. S2.

Figure 2. Comparison of DHβE-bound and nicotine-bound structures of Ls-AChBP.

(a,b) Comparison of the ligand-binding site of the DHβE-bound structure (a, red) with the nicotine-bound structure (b, green). DHβE and nicotine are colored in cyan and purple, respectively. Hydrogen bonds between ligand and its surroundings are shown as stippled lines. The location of the residues is identical except for the residues from the C-loop (residues 185–192). Also, the conformation of the Met114 side chain from the complementary side is different between the two structures. (c) Conformational change of the C-loop due to DHβE binding to Ls-AChBP. The DHβE-bound structure (red) has been superimposed onto the nicotine-bound Ls-AChBP structure (green). (d) The projection vectors belonging to the nicotine-bound and DHβE-bound Ls-AChBP structures are shown in green and red, respectively. The angle between the two projection vectors is 21.4°. Angles between projection vectors of Ls-AChBP co-crystallized with nAChR agonists are listed in Table 2 for comparison. For further details, see Fig. S3.