Dataset Information

Identifying key amino acid residues that affect ?-conotoxin AuIB inhibition of ?3?4 nicotinic acetylcholine receptors.

ABSTRACT: ?-Conotoxin AuIB is a selective ?3?4 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 ?3?4 nAChR, homology modeling, and molecular dynamics simulations to identify the structural determinants of the AuIB·?3?4 nAChR interaction. Two alanine-substituted AuIB analogues, [P6A]AuIB and [F9A]AuIB, did not inhibit the ?3?4 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 ?3?4 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.

SUBMITTER: Grishin AA

PROVIDER: S-EPMC3843058 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Identifying key amino acid residues that affect α-conotoxin AuIB inhibition of α3β4 nicotinic acetylcholine receptors.

Grishin Anton A AA Cuny Hartmut H Hung Andrew A Clark Richard J RJ Brust Andreas A Akondi Kalyana K Alewood Paul F PF Craik David J DJ Adams David J DJ

The Journal of biological chemistry 20131007 48

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 determinant ...[more]

PMID: 24100032

Similar Datasets

Project description:α-Conotoxins (α-CTxs) are small disulfide-rich peptides from venom of Conus species that target nicotinic acetylcholine receptors (nAChRs). The muscle-type nAChRs have been recognized as a potential target for several diseases, such as myogenic disorders, muscle dystrophies, and myasthenia gravis. EI, an α4/7-CTx, mainly blocks α1β1δε nAChRs and has an extra N-terminal extension of three amino acids. In this study, the alanine scanning (Ala-scan) mutagenesis was applied in order to identify key residues of EI for binding with mouse α1β1δε nAChR. The Ala-substituted analogues were tested for their abilities of modulating muscle and neuronal nAChRs in Xenopus laevis oocytes using two-electrode voltage clamp (TEVC) recordings. Electrophysiological results indicated that the vital residues for functional activity of EI were His-7, Pro-8, Met-12, and Pro-15. These changes exhibited a significant decrease in potency of EI against mouse α1β1δε nAChR. Interestingly, replacing the critical serine (Ser) at position 13 with an alanine (Ala) residue resulted in a 2-fold increase in potency at the α1β1δε nAChR, and showed loss of activity on α3β2 and α3β4 nAChRs. Selectivity and potency of [S13A] EI was improved compared with wild-type EI (WT EI). In addition, the structure-activity relationship (SAR) of EI revealed that the "Arg1-Asn2-Hyp3" residues at the N-terminus conferred potency at the muscle-type nAChRs, and the deletion analogue △1-3 EI caused a total loss of activity at the α1β1δε nAChR. Circular dichroism (CD) spectroscopy studies demonstrated that activity loss of truncated analogue △1-3 EI for α1β1δε nAChR is attributed to disturbance of the secondary structure. In this report, an Ala-scan mutagenesis strategy is presented to identify crucial residues that are significantly affecting potency of E1 for mouse α1β1δε nAChR. It may also be important in remodeling of some novel ligands for inhibiting muscle-type nAChRs.

Dataset Information

Identifying key amino acid residues that affect ?-conotoxin AuIB inhibition of ?3?4 nicotinic acetylcholine receptors.

Publications

Identifying key amino acid residues that affect α-conotoxin AuIB inhibition of α3β4 nicotinic acetylcholine receptors.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets