NMR solution conformation of an antitoxic analogue of α-conotoxin GI:: Identification of a common nicotinic acetylcholine receptor α1-subunit binding surface for small ligands and α-conotoxins

The three-dimensional solution conformation of an 11-residue antitoxic analogue of alpha-conotoxin GI, des-Glu1- [Cys3Ala]-des-Cys 13-conotoxin GI (CANPACGRHYS-NH2, designated "GI-15" henceforth), has been determined using two-dimensional H-1 NMR spectroscopy. The disulfide loop region (1C-6C) and the C-terminal tail (8R-11S) are connected by a flexible hinge formed near 7G, and the pairwise backbone rmsds for the former and the latter are 0.58 and 0.65 Angstrom, respectively. Superpositioning GI-15 with the structure of cl-conotoxin GI shows that the two share an essentially identical fold in the common first disulfide loop region (1C-6C). However, the absence of the second disulfide loop in GI-15 results in segmental motion of the C-terminal half, causing the key receptor subtype selectivity residue 8R (Arg9 in alpha-conotoxin GI) to lose its native spatial orientation. The combined features of structural equivalence in the disulfide loop and a mobile C-terminal tail appear to be responsible for the activity of GI-15 as a competitive antagonist against native toxin. Electrostatic surface potential comparisons of the first disulfide region of GI-15 with other alpha-conotoxins or receptor-bound states of acetylcholine and d-tubocurarine show a common protruding surface that may serve as the minimal binding determinant for the neuromuscular acetylcholine receptor alpha(1)-subunit. On the basis of the original "Conus toxin macrosite model" [Olivera, B. M., Rivier, J., Scott, J. K., Hillyard, D. R., and Cruz, L. J. (1991) J. Biol. Chern. 266, 1923-1936], we propose a revised binding model which incorporates these results.