Pairwise electrostatic interactions between alpha-neurotoxins and gamma, delta, and epsilon subunits of the nicotinic acetylcholine receptor

alpha-Neurotoxins bind with high affinity to alpha-gamma and alpha-delta su bunit interfaces of the nicotinic acetylcholine receptor. Since this high a ffinity complex likely involves a van der Waals surface area of similar to 1200 Angstrom(2) and 25-35 residues on the receptor surface, analysis of si de chains should delineate major interactions and the orientation of bound alpha-neurotoxin, Three distinct regions on the gamma subunit, defined by T rp(55), Leu(119), Asp(174), and Glu(176), contribute to alpha-toxin affinit y. Of six charge reversal mutations on the three loops of Naja mossambica m ossambica alpha-toxin, Lys(27) --> Glu, Arg(33) --> Glu, and Arg(36) --> Gl u in loop II reduce binding energy substantially, while mutations in loops I and III hare little effect. Paired residues were analyzed by thermodynami c mutant cycles to delineate electrostatic linkages between the six alpha-t oxin charge reversal mutations and three key residues on the gamma subunit. Large coupling energies were found between Arg(33) at the tip of loop II a nd gamma Leu(119) (-5.7 kcal/mol) and between Lys(27) and gamma Glu(176) (- 5.9 kcal/mol). gamma Trp(55) couples strongly to both Arg(33) and Lys(27), whereas gamma Asp(174) couples minimally to charged alpha-toxin residues. A rg(36), despite strong energetic contributions, does not partner with any g amma subunit residues, perhaps indicating its proximity to the alpha subuni t. By analyzing cationic, neutral and anionic residues in the mutant cycles , interactions at gamma 176 and gamma 119 can be distinguished from those a t gamma 55.