Pairwise electrostatic interactions between α-neurotoxins and γ, δ, and ε subunits of the nicotinic acetylcholine receptor

Abstract

α-Neurotoxins bind with high affinity to α-γ and α-δ subunit interfaces of the nicotinic acetylcholine receptor. Since this high affinity complex likely involves a van der Waals surface area of ~1200 Å2 and 25-35 residues on the receptor surface, analysis of side chains should delineate major interactions and the orientation of bound α-neurotoxin. Three distinct regions on the γ subunit, defined by Trp55, Leu119, Asp174, and Glu176, contribute to α-toxin affinity. Of six charge reversal mutations on the three loops of Naja mossambica mossambica α-toxin, Lys27 → Glu, Arg33 → Glu, and Arg36 → Glu in loop II reduce binding energy substantially, while mutations in loops I and III have little effect. Paired residues were analyzed by thermodynamic mutant cycles to delineate electrostatic linkages between the six α-toxin charge reversal mutations and three key residues on the γ subunit. Large coupling energies were found between Arg33 at the tip of loop II and γLeu119 (-5.7 kcal/mol) and between Lys27 and γGlu176 (-5.9 kcal/mol), γTrp55 couples strongly to both Arg33 and Lys27, whereas γAsp174 couples minimally to charged α-toxin residues. Arg36, despite strong energetic contributions, does not partner with any γ subunit residues, perhaps indicating its proximity to the α subunit. By analyzing cationic, neutral and anionic residues in the mutant cycles, interactions at γ176 and γ119 can be distinguished from those at γ55.