Crystal structure of nicotinic acetylcholine receptor homolog AChBP in complex with an α-conotoxin PnIA variant

Abstract

Conotoxins (Ctx) form a large family of peptide toxins from cone snail venoms that act on a broad spectrum of ion channels and receptors. The subgroup alpha-Ctx specifically and selectively binds to subtypes of nicotinic acetylcholine receptors (nAChRs), which are targets for treatment of several neurological disorders. Here we present the structure at a resolution of 2.4 A of alpha-Ctx PnIA (A10L D14K), a potent blocker of the alpha(7)-nAChR, bound with high affinity to acetylcholine binding protein (AChBP), the prototype for the ligand-binding domains of the nAChR superfamily. Alpha-Ctx is buried deep within the ligand-binding site and interacts with residues on both faces of adjacent subunits. The toxin itself does not change conformation, but displaces the C loop of AChBP and induces a rigid-body subunit movement. Knowledge of these contacts could facilitate the rational design of drug leads using the Ctx framework and may lead to compounds with increased receptor subtype selectivity.

Insight in nAChR subtype selectivity from AChBP crystal structures

“…IMI-AcAChBP co-crystal structures [22,38] show that ImI is stabilized in the binding site in the same orientation as PnIA(A10L D14K) albeit through a broader range of interactions [20]. Some of these interactions overlap, but the nature of most of the contacts varies considerably.…”

“…AChBPs from Lymnaea stagnalis [17], Bulinus truncatus [18] and Aplysia californica [19] share only a 20-24% sequence identity with nAChRs ( Figure 1a) but display a striking structural resemblance with the Torpedo nAChR or mouse α1 nAChR protomer (Figure 1b). Nicotinic ligand binding to these AChBPs was assayed through different methods such as radioligand ( 125 I-αBungarotoxin) displacement, surface plasmon resonance [20][21][22], isothermal titration calorimetry [18] or intrinsic fluorescence quenching [19], and all the AChBPs were found to display a pharmacological profile close to that of the homopentameric α 7 nAChR [18,23].…”

“…A point mutation in the toxin, resulting in PnIA(A10L) results in a complete switch in the selectivity of this toxin towards the α 7 nAChR [48,49]. The PnIA(A10L D14K) variant displays a higher affinity towards LsAChBP and a 3-fold increased binding affinity for the chick α 7 nAChR, but does not display a significant difference in binding with the human [20]. Docking studies performed on the α 7 nAChR further indicate that a series of nAChR specific residues could also affect ligand binding selectivity and affinity [20].…”

Epibatidine binds to four sites on the Torpedo nicotinic acetylcholine receptor

“…The specific subset of residues with which different ligands interact may overlap but are not necessary identical, as illustrated in the crystal structures of the Aplysia AChBP complexed with ligands [6,7]. Further, our kinetic studies [10,11] suggested that each high affinity binding site is associated with a secondary site lying approximately 15Å away.…”

“…The location of these sites has been corroborated by elucidation of the crystal structures of related acetylcholine binding proteins (AChBPs) [e.g. [5][6][7]. There is, however, a body of information to indicate that the binding sites may be more complex than is readily apparent *Corresponding author.…”