The mammalian skeletal muscle acetylcholine receptor contains two nonequivalent acetylcholine binding sites, one each at the alpha/delta and alpha/gamma subunit interfaces. Alpha-Conotoxin MI, a 14-amino acid competitive antagonist, binds at both interfaces but has approximately 10(4) higher affinity for the alpha/delta site. We performed an “alanine walk” to identify the residues in alpha-MI that contribute to this selective interaction with the alpha/delta site. Electrophysiological measurements with Xenopus oocytes expressing normal receptors or receptors lacking either the gamma or delta subunit were made to assay toxin-receptor interaction. Alanine substitutions in most amino acid positions had only modest effects on toxin potency at either binding site. However, substitutions in two positions, proline-6 and tyrosine-12, dramatically reduced toxin potency at the high-affinity alpha/delta site while having comparatively little effect on low-affinity alpha/gamma binding. When tyrosine-12 was replaced by alanine, the toxin’s selectivity for the high-affinity site (relative to that for the low-affinity site) was reduced from 45,000- to 30-fold. A series of additional amino acid substitutions in this position showed that increasing side chain size/hydrophobicity increases toxin potency at the alpha/delta site without affecting alpha/gamma binding. In contrast, when tyrosine-12 is diiodinated, toxin binding is nearly irreversible at the alpha/delta site but also increases by approximately 500-fold at the alpha/gamma site. The effects of position 12 substitutions are accounted for almost entirely by changes in the rate of toxin dissociation from the high-affinity alpha/delta binding site.

Jacobsen, R.B., et al. (1999) Critical residues influence the affinity and selectivity of alpha-conotoxin MI for nicotinic acetylcholine receptors, Biochemistry. PMID: 10529206

The toxic peptide from Conus magus venom (conotoxin MI) is a 14-amino acid peptide (McIntosh, M., Cruz, L. J., Hunkapiller, M. W., Gray, W. R., and Olivera, B. M. (1982) Arch. Biochem. Biophys. 218, 329-334) which inhibits the acetylcholine receptor. In this work we have confirmed the primary structure and established the disulfide bonding configuration (Cys 3-Cys 8; Cys 4-Cys 14) by direct chemical synthesis of the toxin with specific disulfide bridges. Natural and synthetic toxins were compared by several methods. Fast atom bombardment mass spectroscopy confirmed that the synthetic product had the expected molecular mass and number of exchangeable hydrogens. Ultraviolet CD spectra were closely comparable in shape and magnitude for the two materials, which were also identical in biological activity and chromatographic behavior. We have also established that, although the peptide is highly cross-linked with two disulfide bridges, it can slowly equilibrate between two conformations. A simulation analysis suggests that the conformers have half-lives of approximately 12 and approximately 72 min at 0 degrees C, decreasing approximately 2-fold for every 10 degrees C increase in temperature.

Gray, W. R., et al. (1983) Conotoxin MI. Disulfide bonding and conformational states, J Biol Chem. PMID: 6630187

We have tested the importance of charge interactions for alpha-conotoxin MI binding to the nicotinic acetylcholine receptor (AChR). Ionic residues on alpha-conotoxin MI were altered by site-directed mutagenesis or by chemical modification. In physiological buffer, removal of charges at the N terminus, His-5, and Lys-10 had small (2-4-fold) effects on binding affinity to the mouse muscle AChR and the Torpedo AChR. It was also demonstrated that conotoxin had no effect on the conformational equilibrium of either receptor, as assessed by the effects of the noncompetitive antagonist proadifen on conotoxin binding and, conversely, the effect of conotoxin on the affinity of phencyclidine, proadifen, and ethidium. Conotoxin displayed higher binding affinity in low ionic strength buffer; neutralization of Lys-10 and the N terminus by acetylation blocked this affinity shift at the alphadelta site but not at the alphagamma site. It is concluded that Ctx residues Lys-10 and the N terminal interact with oppositely charged receptor residues only at the alphadelta site, and the two sites have distinct arrangements of charged residues. Ethidium fluorescence experiments demonstrated that conotoxin is formally competitive with a small cholinergic ligand, tetramethylammonium. Thus, alpha-conotoxin MI appears to interact with the portion of the binding site responsible for stabilizing agonist cations but does not do so with a cationic residue and is, consequently, incapable of inducing a conformational change.

Papineni RV., et al. (2001) Site-specific charge interactions of alpha-conotoxin MI with the nicotinic acetylcholine receptor. J Biol Chem. PMID: 11323431