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

Anton A Grishin,Hartmut Cuny,Andrew Hung,Richard J Clark,Andreas Brust,Kalyana Akondi,Paul F Alewood,David J Craik,David J Adams

doi:10.1074/jbc.m113.512582

Anton A Grishin, Hartmut Cuny + Show 7 more

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https://doi.org/10.1074/jbc.m113.512582

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Abstract

α-Conotoxin AuIB is a selective α3β4 nicotinic acetylcholine receptor (nAChR) subtype inhibitor. Its analgesic properties are believed to result from it activating GABAB receptors and subsequently inhibiting CaV2.2 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 α3β4 nAChR, homology modeling, and molecular dynamics simulations to identify the structural determinants of the AuIB·α3β4 nAChR interaction. Two alanine-substituted AuIB analogues, [P6A]AuIB and [F9A]AuIB, did not inhibit the α3β4 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 α3β4 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 β4 nAChR subunit. This hypothesis was confirmed by site-directed mutagenesis of the β4-Trp-59 and β4-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 β4 subunit and gave further insight into how AuIB Phe-9 interacts with the β4 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.

Highlights

Peptides isolated from the venom of cone snails belonging to the genus Conus are valuable pharmacological tools, and some are promising drug leads [1,2,3,4,5]. ␣-Conotoxins are a subfamily of these peptides and consist of 12–19 amino acid residues, including four cysteines with a characteristic CC-C-C arrangement [6, 7]
Two other ␣-conotoxins, BuIA, an unusual 4/4-␣-conotoxin isolated from the venom of Conus bullatus, and the recently described 4/7-␣-conotoxin RegIIA from Conus regius, have been found to be considerably more potent inhibitors of the ␣3␤4 subtype, blocking it at low nanomolar concentrations [16, 17]
Alanine Scanning Mutagenesis Identifies Residues in the AuIB Sequence That Are Critical for Its Interaction with the ␣3␤4 Nicotinic acetylcholine receptors (nAChRs) Subtype—To find residues in the AuIB sequence that contribute most to ␣3␤4 nAChR inhibition, we performed alanine scanning mutagenesis of the peptide

Summary

Introduction

Peptides isolated from the venom of cone snails belonging to the genus Conus are valuable pharmacological tools, and some are promising drug leads [1,2,3,4,5]. ␣-Conotoxins are a subfamily of these peptides and consist of 12–19 amino acid residues, including four cysteines with a characteristic CC-C-C arrangement (type I cysteine framework) [6, 7]. Homology Modeling and MD Simulations Suggest Loss of Interactions between AuIB and Key Receptor Residues When Ala Is Substituted for Phe-9—Having established that position 9 Phe in AuIB is crucial for the peptide inhibiting ␣3␤4, we used atomistic simulations of native AuIB and the [F9A]AuIB mutant bound to ␣3␤4 to provide molecular-level explanations of why the mutation so markedly reduces inhibition.

Results

Conclusion