Identification of pairwise interactions in the α-neurotoxin-nicotinic acetylcholine receptor complex through double mutant cycles

alpha-Neurotoxins are potent inhibitors of the nicotinic acetylcholine receptor (nAChR), binding with high affinity to the two agonist sites located on the extracellular domain. Previous site-directed mutagenesis had identified three residues on the Lu-neurotoxin from Naja mossambica mossambica (Lys(27), Arg(33), and Lys(47)), and four residues on the mouse muscle nAChR cy-subunit (Val(188), Tyr(190), Pro(197) and Asp(200)) as contributing to binding, In this study, thermodynamic mutant cycle analysis was applied to these sets of residues to identify specific pairwise interactions. Amino acid variants of alpha-neurotoxin from N., mossambica mossambica at position 33 and of the nAChR at position 188 showed strong energetic couplings of 2-3 kcal/mol at both binding sites. Consistently smaller yet significant linkages of 1.6-2.1 kcal/mol were also observed between variants at position 27 on the toxin and position 188 on the receptor. Additionally, toxin residue 27 coupled to the receptor residues 190, 197, and 200 at the alpha delta binding site with observed coupling energies of 1.5-1.9 kcal/mol, No Linkages were found between toxin residue Lys(47) and the receptor residues studied here, These results provide direct evidence that the two conserved cationic residues Arg(33) and Lys(27), located on loop II of the toxin structure, are binding in close proximity to the alpha-subunit region between residues 188-200. The toxin residue Arg(33) is closer to Val(188), where it is likely stabilized by adjacent negative or aromatic residues on the receptor structure. Lys(27) is positioned closer to Tyr(190), Pro(197) and Asp(200), where it is likely stabilized through electrostatic interaction with Asp(200) and/or cation/pi; interactions with Try(190).