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

The Journal of Biological Chemistry
E J AckermannP Taylor

Abstract

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 alpha-neurotoxin from Naja mossambica mossambica (Lys27, Arg33, and Lys47) and four residues on the mouse muscle nAChR alpha-subunit (Val188, Tyr190, Pro197, and Asp200) 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 alphadelta binding site with observed coupling energies of 1.5-1.9 kcal/mol. No linkages were found between toxin residue Lys47 and the receptor residues studied here. These results provide direct evidence that the two con...Continue Reading

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