Waglerin-1 (Wtx-1) is a 22-amino acid peptide that competitively antagonizes muscle nicotinic acetylcholine receptors (nAChRs). Previous work demonstrated that Wtx-1 binds to mouse nAChRs with higher affinity than receptors from rats or humans, and distinguished residues in α and ε subunits that govern the species selectivity. These studies also showed that Wtx-1 binds selectively to the α-ε binding site with significantly higher affinity than to the α-ε binding site. Here we identify residues at equivalent positions in the ε, γ, and δ subunits that govern Wtx-1 selectivity for one of the two binding sites on the nAChR pentamer. Using a series of chimeric and point mutant subunits, we show that residues Gly-57, Asp-59, Tyr-111, Tyr-115, and Asp-173 of the ε subunit account predominantly for the 3700-fold higher affinity of the α-ε site relative to that of the α-γ site. Similarly, we find that residues Lys-34, Gly-57, Asp-59, and Asp-173 account predominantly for the high affinity of the α-ε site relative to that of the α-δ site. Analysis of combinations of point mutations reveals that Asp-173 in the ε subunit is required together with the remaining determinants in the ε subunit to achieve Wtx-1 selectivity. In particular, Lys-34 interacts with Asp-173 to confer high affinity, resulting in a ΔΔGINT of -2.3 kcal/mol in the ∈ subunit and a ΔΔGINT of -1.3 kcal/mol in the δ subunit, Asp-173 is part of a nonhomologous insertion not found in the acetylcholine binding protein structure. The key role of this insertion in Wtx-1 selectivity indicates that it is proximal to the ligand binding site. We use the binding and interaction energies for Wtx-1 to generate structural models of the α-ε,α-γ, and α-δ binding sites containing the nonhomologous insertion.
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