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

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.