Identifying Key Amino Acid Residues That Affect α-Conotoxin AuIB Inhibition of α3β4 Nicotinic Acetylcholine Receptors

Background: -Conotoxin AuIB interacts with 34 nAChRs and GABA(B) receptors, but structural determinants of these interactions are unknown. Results: Using alanine scanning mutagenesis and molecular dynamics, we identified residues crucial for AuIB34 nAChR interaction. Conclusion: We identified the key residues that mediate AuIB34 nAChR interaction. Significance: Ability to direct -conotoxin binding to nAChRs or GABA(B) receptors will improve analgesic conopeptides. -Conotoxin AuIB is a selective 34 nicotinic acetylcholine receptor (nAChR) subtype inhibitor. Its analgesic properties are believed to result from it activating GABA(B) receptors and subsequently inhibiting Ca(V)2.2 voltage-gated calcium channels. The structural determinants that mediate diverging AuIB activity at these targets are unknown. We performed alanine scanning mutagenesis of AuIB and 34 nAChR, homology modeling, and molecular dynamics simulations to identify the structural determinants of the AuIB34 nAChR interaction. Two alanine-substituted AuIB analogues, [P6A]AuIB and [F9A]AuIB, did not inhibit the 34 nAChR. NMR and CD spectroscopy studies demonstrated that [F9A]AuIB retains its native globular structure, so its activity loss is probably due to loss of specific toxin-receptor residue pairwise contacts. Compared with AuIB, the concentration-response curve for inhibition of 34 by [F9A]AuIB shifted rightward more than 10-fold, and its subtype selectivity profile changed. Homology modeling and molecular dynamics simulations suggest that Phe-9 of AuIB interacts with a two-residue binding pocket on the 4 nAChR subunit. This hypothesis was confirmed by site-directed mutagenesis of the 4-Trp-59 and 4-Lys-61 residues of loop D, which form a putative binding pocket. AuIB analogues with Phe-9 substitutions corroborated the finding of a binding pocket on the 4 subunit and gave further insight into how AuIB Phe-9 interacts with the 4 subunit. In summary, we identified critical residues that mediate interactions between AuIB and its cognate nAChR subtype. These findings might help improve the design of analgesic conopeptides that selectively avoid nAChR receptors while targeting receptors involved with nociception.