Abstract
The present work uses alpha-conotoxin ImI (CTx ImI) to probe the neurotransmitter binding site of neuronal alpha7 acetylcholine receptors. We identify key residues in alpha7 that contribute to CTx ImI affinity, and use mutant cycles analysis to identify pairs of residues that stabilize the receptor-conotoxin complex. We first mutated key residues in the seven known loops of alpha7 that converge at the subunit interface to form the ligand binding site. The mutant subunits were expressed in 293 HEK cells, and CTx ImI binding was measured by competition against the initial rate of 125I-alpha-bungarotoxin binding. The results reveal a predominant contribution by Tyr-195 in alpha7, accompanied by smaller contributions by Thr-77, Tyr-93, Asn-111, Gln-117, and Trp-149. Based upon our previous identification of bioactive residues in CTx ImI, we measured binding of receptor and toxin mutations and analyzed the results using thermodynamic mutant cycles. The results reveal a single dominant interaction between Arg-7 of CTx ImI and Tyr-195 of alpha7 that anchors the toxin to the binding site. We also find multiple weak interactions between Asp-5 of CTx ImI and Trp-149, Tyr-151, and Gly-153 of alpha7, and between Trp-10 of CTx ImI and Thr-77 and Asn-111 of alpha7. The overall results establish the orientation of CTx ImI as it bridges the subunit interface and demonstrate close approach of residues on opposing faces of the alpha7 binding site.
Highlights
Recent studies have used small protein toxins to probe active sites of membrane proteins from excitable cells
We find that Tyr-195 in loop C provides the dominant source of stabilization of conotoxin ImI (CTx ImI), as shown by the 320-fold loss in affinity produced by Y195T (Fig. 3 and Table I)
In addition to Tyr-195, we find that residues in loops A, B, and C contribute to CTx ImI binding, showing 10- to 30-fold changes in affinity for the various mutations; these include Y93T, W149T, Y151T, G153S, R186V, and D197N (Fig. 3 and Table I)
Summary
Materials—125I-Labeled ␣-bungarotoxin (␣-Bgt) was purchased from NEN Life Science Products, d-tubocurarine chloride from ICN Pharmaceuticals, 293 human embryonic kidney cell line (293 HEK) from the American Type Culture Collection, and unlabeled ␣-Bgt from Sigma Chemicals. The two intramolecular disulfide bridges were formed as follows; the S-triphenylmethyl protecting groups attached to cysteines 3 and 12 were removed during trifluoroacetic acid cleavage of the linear peptide from the support resin. The peptide was oxidized by molecular oxygen to form the 3–12 disulfide bond by stirring in 50 mM ammonium bicarbonate buffer, pH 8.5, at 25 °C for 24 h. The peptide was lyophilized prior to formation of the second disulfide bond. The acetamidomethyl-protecting groups on cysteine 2 and 8 were removed oxidatively by iodine as described [17], except the peptide/iodine reaction was allowed to progress for 16 h prior to carbon tetrachloride extraction. Mutant cDNAs were constructed by bridging naturally occurring or mutagenically installed restriction sites with doublestranded oligonucleotides or by the Quick ChangeTM site-directed mutagenesis kit (Stratagene).
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