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Periplasmic Expression of 4/7 ?-Conotoxin TxIA Analogs in E. coli Favors Ribbon Isomer Formation - Suggestion of a Binding Mode at the ?7 nAChR.

ABSTRACT: Peptides derived from animal venoms provide important research tools for biochemical and pharmacological characterization of receptors, ion channels, and transporters. Some venom peptides have been developed into drugs (such as the synthetic ?-conotoxin MVIIA, ziconotide) and several are currently undergoing clinical trials for various clinical indications. Challenges in the development of peptides include their usually limited supply from natural sources, cost-intensive chemical synthesis, and potentially complicated stereoselective disulfide-bond formation in the case of disulfide-rich peptides. In particular, if extended structure-function analysis is performed or incorporation of stable isotopes for NMR studies is required, the comparatively low yields and high costs of synthesized peptides might constitute a limiting factor. Here we investigated the expression of the 4/7 ?-conotoxin TxIA, a potent blocker at ?3?2 and ?7 nicotinic acetylcholine receptors (nAChRs), and three analogs in the form of maltose binding protein fusion proteins in Escherichia coli. Upon purification via nickel affinity chromatography and release of the toxins by protease cleavage, HPLC analysis revealed one major peak with the correct mass for all peptides. The final yield was 1-2 mg of recombinant peptide per liter of bacterial culture. Two-electrode voltage clamp analysis on oocyte-expressed nAChR subtypes demonstrated the functionality of these peptides but also revealed a 30 to 100-fold potency decrease of expressed TxIA compared to chemically synthesized TxIA. NMR spectroscopy analysis of TxIA and two of its analogs confirmed that the decreased activity was due to an alternative disulfide linkage rather than the missing C-terminal amidation, a post-translational modification that is common in ?-conotoxins. All peptides preferentially formed in the ribbon conformation rather than the native globular conformation. Interestingly, in the case of the ?7 nAChR, but not the ?3?2 subtype, the loss of potency could be rescued by an R5D substitution. In conclusion, we demonstrate efficient expression of functional but alternatively folded ribbon TxIA variants in E. coli and provide the first structure-function analysis for a ribbon 4/7-?-conotoxin at ?7 and ?3?2 nAChRs. Computational analysis based on these data provide evidence for a ribbon ?-conotoxin binding mode that might be exploited to design ligands with optimized selectivity.

SUBMITTER: El Hamdaoui Y

PROVIDER: S-EPMC6554660 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Periplasmic Expression of 4/7 α-Conotoxin TxIA Analogs in <i>E. coli</i> Favors Ribbon Isomer Formation - Suggestion of a Binding Mode at the α7 nAChR.

El Hamdaoui Yamina Y Wu Xiaosa X Clark Richard J RJ Giribaldi Julien J Anangi Raveendra R Craik David J DJ King Glenn F GF Dutertre Sebastien S Kaas Quentin Q Herzig Volker V Nicke Annette A

Frontiers in pharmacology 20190531

Peptides derived from animal venoms provide important research tools for biochemical and pharmacological characterization of receptors, ion channels, and transporters. Some venom peptides have been developed into drugs (such as the synthetic ω-conotoxin MVIIA, ziconotide) and several are currently undergoing clinical trials for various clinical indications. Challenges in the development of peptides include their usually limited supply from natural sources, cost-intensive chemical synthesis, and ...[more]

PMID: 31214027

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Project description:α-Conotoxins potently inhibit isoforms of nicotinic acetylcholine receptors (nAChRs), which are essential for neuronal and neuromuscular transmission. They are also used as neurochemical tools to study nAChR physiology and are being evaluated as drug leads to treat various neuronal disorders. A number of experimental studies have been performed to investigate the structure-activity relationships of conotoxin/nAChR complexes. However, the structural determinants of their binding interactions are still ambiguous in the absence of experimental structures of conotoxin-receptor complexes. In this study, the binding modes of α-conotoxin ImI to the α7-nAChR, currently the best-studied system experimentally, were investigated using comparative modeling and molecular dynamics simulations. The structures of more than 30 single point mutants of either the conotoxin or the receptor were modeled and analyzed. The models were used to explain qualitatively the change of affinities measured experimentally, including some nAChR positions located outside the binding site. Mutational energies were calculated using different methods that combine a conformational refinement procedure (minimization with a distance dependent dielectric constant or explicit water, or molecular dynamics using five restraint strategies) and a binding energy function (MM-GB/SA or MM-PB/SA). The protocol using explicit water energy minimization and MM-GB/SA gave the best correlations with experimental binding affinities, with an R2 value of 0.74. The van der Waals and non-polar desolvation components were found to be the main driving force for binding of the conotoxin to the nAChR. The electrostatic component was responsible for the selectivity of the various ImI mutants. Overall, this study provides novel insights into the binding mechanism of α-conotoxins to nAChRs and the methodological developments reported here open avenues for computational scanning studies of a rapidly expanding range of wild-type and chemically modified α-conotoxins.

Dataset Information

Periplasmic Expression of 4/7 ?-Conotoxin TxIA Analogs in E. coli Favors Ribbon Isomer Formation - Suggestion of a Binding Mode at the ?7 nAChR.

Publications

Periplasmic Expression of 4/7 α-Conotoxin TxIA Analogs in <i>E. coli</i> Favors Ribbon Isomer Formation - Suggestion of a Binding Mode at the α7 nAChR.

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