Hydrophobic pairwise interactions stabilize α-conotoxin MI in the muscle acetylcholine receptor binding site

Abstract

The present work delineates pairwise interactions underlying the nanomolar affinity of α-conotoxin MI (CTx MI) for the α-δ site of the muscle acetylcholine receptor (AChR). We mutated all non-cysteine residues in CTx MI, expressed the α2βδ2 pentameric form of the AChR in 293 human embryonic kidney cells, and measured binding of the mutant toxins by competition against the initial rate of 125I-α-bungarotoxin binding. The CTx MI mutations P6G, A7V, G9S, and Y12T all decrease affinity for α2βδ2 pentamers by 10,000-fold. Side chains at these four positions localize to a restricted region of the known three-dimensional structure of CTx MI. Mutations of the AChR reveal major contributions to CTx MI affinity by Tyr- 198 in the a subunit and by the selectivity determinants Ser-36, Tyr-113, and Ile-178 in the δ subunit. By using double mutant cycles analysis, we find that Tyr-12 of CTx MI interacts strongly with all three selectivity determinants in the δ subunit and that δSer-36 and δIle-178 are interdependent in stabilizing Tyr-12. We find additional strong interactions between Gly-9 and Pro-6 in CTx MI and selectivity determinants in the δ subunit, and between Ala-7 and Pro-6 and Tyr-198 in the α subunit. The overall results reveal the orientation of CTx MI when bound to the α-δ interface and show that primarily hydrophobic interactions stabilize the complex.