Residues in the ε Subunit of the Nicotinic Acetylcholine Receptor Interact To Confer Selectivity of Waglerin-1 for the α−ε Subunit Interface Site

Abstract

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 alpha and epsilon subunits that govern the species selectivity. These studies also showed that Wtx-1 binds selectively to the alpha-epsilon binding site with significantly higher affinity than to the alpha-delta binding site. Here we identify residues at equivalent positions in the epsilon, gamma, and delta 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 epsilon subunit account predominantly for the 3700-fold higher affinity of the alpha-epsilon site relative to that of the alpha-gamma site. Similarly, we find that residues Lys-34, Gly-57, Asp-59, and Asp-173 account predominantly for the high affinity of the alpha-epsilon site relative to that of the alpha-delta site. Analysis of combinations of point mutations reveals that Asp-173 in the epsilon subunit is required together with the remaining determinants in the epsilon subunit to achieve Wtx-1 selectivity. In particular, Lys-34 interacts with Asp-173 to confer high affinity, resulting in a DeltaDeltaG(INT) of -2.3 kcal/mol in the epsilon subunit and a DeltaDeltaG(INT) of -1.3 kcal/mol in the delta 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 alpha-epsilon, alpha-gamma, and alpha-delta binding sites containing the nonhomologous insertion.

Structural Basis for Epibatidine Selectivity at Desensitized Nicotinic Receptors

“…We hypothesize that because the template upon which our model is based, AChBP, has been suggested to have been crystallized in a desensitized-like conformation (Fruchart-Gaillard et al, 2002), our computationally derived structure should provide useful information about binding site structure in the desensitized state. Consistent with previous structural models of the receptor (Le Molles et al, 2002b;Sine et al, 2002;Chiara et al, 2003;Le Novere, 2004), the agonist binding sites in our model are formed by the convergence of a series of mostly aromatic residues from the ␣-subunit (Tyr93, Trp149, Tyr190, Cys192, Cys193, and Tyr198) and a series of complementary residues from the non-␣-subunit. In our epibatidine-docked complex, this non-␣ contribution comprises several residues contained within an extended hairpin structure, formed by residues ␥Asn94 -Ser127, which passes diagonally through the extracellular domain of the subunit.…”

“…The final epibatidine determinant in this region, ␥Tyr117/␦Thr119, was also identified as a major determinant of metocurine selectivity (Sine, 1993) and has been suggested to make direct contact with this competitive antagonist (Fu and Sine, 1994;Gao et al, 2003). Also within this region are ␥Leu109/␦Leu111, which was recently shown to be labeled by the competitive antagonist 4-[(3-trifluoromethyl)-3H-diazirin-3-yl]benzoylcholine (Chiara et al, 2003), ␥Cys115/␦Tyr117, which contributes to carbamylcholine (Prince and Sine, 1996) and Waglerin (Molles et al, 2002b) selectivity, and ␥Ile116/␦Val118, which contributes to metocurine selectivity (Sine, 1993). Overall, our present results, combined with findings from previous studies, demonstrate a major role for ␥104 -117/␦106 -119 in conferring ligand binding selectivity at the nAChR.…”

“…Previous studies in our laboratories probed the structure of the nAChR using chimeric subunits to identify amino acid differences between ␥ and ␦ that determine agonist and antagonist selectivity in the resting, activatible state of the receptor (Sine, 1993(Sine, , 1997Bren and Sine, 1997;Prince and Sine, 1996;Molles et al, 2002b). The emerging overall picture showed that four loops from the ␥-and ␦-subunits (loops D-G), well separated along the primary sequence, contribute to each binding site interface.…”

β2 Subunit Contribution to 4/7 α-Conotoxin Binding to the Nicotinic Acetylcholine Receptor

“…Docking simulations with ImI and PnIB structures on an ␣7 nAChR model revealed overlapping but different binding sites for ␣-conotoxins and snake toxins (17). Both ␣-conotoxins block the receptor by targeting a small cleft above the ␤9/10 hairpin that is also blocked by the small peptide antagonist toxin waglerin (25) and the non-peptidic antagonist d-tubocurarine (26). Concerning the ␣3␤2 nAChR, only one residue (␤2-Thr-57) weakly affecting 4/7 conotoxin binding has been identified on the ␤2 subunit compared with four on the ␣3 subunit (27,28).…”

Probing the Reorganization of the Nicotinic Acetylcholine Receptor during Desensitization by Time-Resolved Covalent Labeling Using [3H]AC5, a Photoactivatable Agonist

“…7.0; Tripos, St. Louis, MO) was used to mutate the nAChR sequences into the AChBP structure. Because the most important requirement in homology modeling is a correct alignment between the sequence to be modeled with that of the template structure, we compared the alignment proposed by Brejc et al (2001) with other alignments that have been published recently (Le Novere et al, 2002;Molles et al, 2002;Schapira et al, 2002). All multiple alignments show a good overall conservation within the secondary structure and the different loops, except for the F segment.…”