Identifying key amino acid residues that affect alpha-conotoxin AuIB inhibition of alpha3beta4 nicotinic acetylcholine receptors

Grishin, Anton A., Cuny, Hartmut, Hung, Andrew, Clark, Richard J., Brust, Andreas, Akondi, Kalyana, Alewood, Paul F., Craik, David J. and Adams, David J. (2013) Identifying key amino acid residues that affect alpha-conotoxin AuIB inhibition of alpha3beta4 nicotinic acetylcholine receptors. Journal of Biological Chemistry, 288 48: 34428-34442. doi:10.1074/jbc.M113.512582

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Author Grishin, Anton A.
Cuny, Hartmut
Hung, Andrew
Clark, Richard J.
Brust, Andreas
Akondi, Kalyana
Alewood, Paul F.
Craik, David J.
Adams, David J.
Title Identifying key amino acid residues that affect alpha-conotoxin AuIB inhibition of alpha3beta4 nicotinic acetylcholine receptors
Formatted title
Identifying key amino acid residues that affect α-conotoxin AuIB inhibition of α3β4 nicotinic acetylcholine receptors
Journal name Journal of Biological Chemistry   Check publisher's open access policy
ISSN 0021-9258
1083-351X
Publication date 2013-11-29
Year available 2013
Sub-type Article (original research)
DOI 10.1074/jbc.M113.512582
Open Access Status File (Publisher version)
Volume 288
Issue 48
Start page 34428
End page 34442
Total pages 27
Place of publication Bethesda, MD, United States
Publisher American Society for Biochemistry and Molecular Biology
Language eng
Abstract 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.
Formatted 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 ten-fold and its subtype selectivity profile changed. Homology modeling and molecular dynamics simulations suggest that Phe9 of AuIB interacts with a two-residue binding pocket on the β4 nAChR subunit. This hypothesis was confirmed by site-directed mutagenesis of the Trp59-β4 and Lys61-β4 residues of loop D, which formed a putative binding pocket. AuIB analogues with Phe9 substitutions corroborated the finding of a binding pocket on the β4 subunit and gave further insight into how AuIB Phe9 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.

Background: α-Conotoxin AuIB interacts with α3β4 nAChRs and GABABRs, but structural determinants of these interactions are unknown.

Results: Using alanine scanning mutagenesis and molecular dynamics, we identified residues crucial for AuIB–α3β4 nAChR interaction.

Conclusions: We identified the key residues that mediate AuIB–α3β4 nAChR interaction.

Significance: Ability to direct α-conotoxin binding to nAChRs or GABABRs will improve analgesic conopeptides.

Keyword Conotoxin
Nicotinic acetylcholine receptor
Electrophysiology
NMR structure
Molecular modeling
Molecular docking
Peptide chemical synthesis
Site-directed mutagenesis
Xenopus oocyte
NMR
Site directed mutagenesis
Q-Index Code C1
Q-Index Status Confirmed Code
Grant ID 569927
DP 1093115
Institutional Status UQ
Additional Notes First Published: 7 October 2013.

Document type: Journal Article
Sub-type: Article (original research)
Collections: Queensland Brain Institute Publications
Official 2014 Collection
Institute for Molecular Bioscience - Publications
 
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Created: Fri, 22 Nov 2013, 01:09:53 EST by Susan Allen on behalf of Institute for Molecular Bioscience