Structure determination of the three disulfide bond isomers of α-conotoxin GI: a model for the role of disulfide bonds in structural stability

Abstract

The three possible disulfide bonded isomers of α-conotoxin GI have been selectively synthesised and their structures determined by 1H NMR spectroscopy. α-Conotoxin GI derives from the venom of Conus geographus and is a useful neuropharmacological tool as it selectively binds to the nicotinic acetylcholine receptor (nAChR), a ligand-gated ion channel involved in nerve signal transmission. The peptide has the sequence ECCNPACGRHYSC-NH2, and the three disulfide bonded isomers are referred to as GI(2–7;3–13), GI(2–13;3–7) and GI(2–3;7–13). The NMR structure for the native isomer GI(2–7;3–13) is of excellent quality, with a backbone pairwise RMSD of 0.16 Å for a family of 35 structures, and comprises primarily a distorted 310helix between residues 5 to 11. The two non-native isomers exhibit multiple conformers in solution, with the major populated forms being different in structure both from each other and from the native form. Structure-activity relationships for the native GI(2–7;3–13) as well as the role of the disulfide bonds on folding and stability of the three isomers are examined. It is concluded that the disulfide bonds in α-conotoxin GI play a crucial part in determining both the structure and stability of the peptide. A trend for increased conformational heterogeneity was observed in the order of GI(2–7;3–13)<GI(2–13;3–7)<GI(2–3;7–13). It was found that the peptide bond joining Cys2 to Cys3 in GI(2–3;7–13) is predominantly trans, rather than cis as theoretically predicted. These structural data are used to interpret the varying nAChR binding of the non-native forms.A model for the binding of native GI(2–7;3–13) to the mammalian nAChR is proposed, with an α-subunit binding face made up of Cys2, Asn4, Pro5, Ala6 and Cys7 and a selectivity face, comprised of Arg9 and His10. These two faces orient the molecule between the α and δ subunits of the receptor. The structure of the CCNPAC sequence of the native GI(2–7;3–13) is compared to the structure of the identical sequence from the toxic domain of heat-stable enterotoxins, which forms part of the receptor binding region of the enterotoxins, but which has a different disulfide connectivity.