Hydrophobic Pairwise Interactions Stabilize α-Conotoxin MI in the Muscle Acetylcholine Receptor Binding Site

Nina Bren,Steven M Sine

doi:10.1074/jbc.275.17.12692

Abstract

The present work delineates pairwise interactions underlying the nanomolar affinity of alpha-conotoxin MI (CTx MI) for the alpha-delta site of the muscle acetylcholine receptor (AChR). We mutated all non-cysteine residues in CTx MI, expressed the alpha(2)betadelta(2) 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 (125)I-alpha-bungarotoxin binding. The CTx MI mutations P6G, A7V, G9S, and Y12T all decrease affinity for alpha(2)betadelta(2) 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 alpha subunit and by the selectivity determinants Ser-36, Tyr-113, and Ile-178 in the delta subunit. By using double mutant cycles analysis, we find that Tyr-12 of CTx MI interacts strongly with all three selectivity determinants in the delta subunit and that deltaSer-36 and deltaIle-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 delta subunit, and between Ala-7 and Pro-6 and Tyr-198 in the alpha subunit. The overall results reveal the orientation of CTx MI when bound to the alpha-delta interface and show that primarily hydrophobic interactions stabilize the complex.

Highlights

Recent studies have used protein toxins to probe active sites of ligand- and voltage-gated ion channels [1,2,3,4,5,6]
Materials—␣-Conotoxin MI was purchased from American Peptide Company; 293 human embryonic kidney cell line (293 HEK) and BOSC 23 HEK cell line were from the American Type Culture Collection; 125I-labeled ␣-bungarotoxin was from NEN Life Science Products; dtubocurarine chloride was from ICN Pharmaceuticals; and 5,5Ј-dithiobis-2-nitrobenzoic acid was from Sigma
Because conotoxin MI (CTx MI) binds 10,000-fold more tightly to the ␣-␦ than to the ␣-␥ interface of the acetylcholine receptor (AChR) [10], we measured CTx MI binding to the ␣2␤␦2 pentameric form of the AChR expressed on the surface of 293 HEK cells

Summary

EXPERIMENTAL PROCEDURES

Materials—␣-Conotoxin MI was purchased from American Peptide Company; 293 human embryonic kidney cell line (293 HEK) and BOSC 23 HEK cell line were from the American Type Culture Collection; 125I-labeled ␣-bungarotoxin was from NEN Life Science Products; dtubocurarine chloride was from ICN Pharmaceuticals; and 5,5Ј-dithiobis-2-nitrobenzoic acid was from Sigma. The double mutant ␦(S36K/␦Y113S) was constructed by ligation of the 400-bp PflMI-PflMI fragment containing ␦Y113S to the 5100-bp PflMI-PflMI fragment containing the mutation S36K. Human embryonic kidney cells (293 HEK) were transfected with mutant or wild type subunit cDNAs using calcium phosphate precipitation as described previously [16]. Ligand Binding Measurements—␣-Conotoxin MI binding to intact cells was measured by competition against the initial rate of 125Ilabeled ␣-bungarotoxin [18]. The cells were briefly centrifuged, resuspended in potassium Ringer’s solution, and divided into aliquots for ␣-conotoxin binding measurements. Binding was terminated by the addition of 2 ml of potassium Ringer’s solution containing 600 ␮M d-tubocurarine chloride. Means Ϯ S.D. of the individual fitted parameters are presented (Tables II and III)

RESULTS

Observed Mr

DISCUSSION

WT CTx MI

TABLE IV Omega values from double mutant cycles analysis