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

El Hamdaoui, Yamina; Wu, Xiaosa; Clark, Richard J.; Giribaldi, Julien; Anangi, Raveendra; Craik, David J.; King, Glenn F.; Dutertre, Sebastien; Kaas, Quentin; Herzig, Volker und Nicke, Annette (31. Mai 2019): Periplasmic Expression of 4/7 α-Conotoxin TxIA Analogs in E. coli Favors Ribbon Isomer Formation – Suggestion of a Binding Mode at the α7 nAChR. In: Frontiers in Pharmacology, Bd. 10, 577: S. 1-17

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 omega-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 alpha-conotoxin TxIA, a potent blocker at alpha 3 beta 2 and alpha 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 alpha-conotoxins. All peptides preferentially formed in the ribbon conformation rather than the native globular conformation. Interestingly, in the case of the alpha 7 nAChR, but not the alpha 3 beta 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-alpha-conotoxin at alpha 7 and alpha 3 beta 2 nAChRs. Computational analysis based on these data provide evidence for a ribbon alpha-conotoxin binding mode that might be exploited to design ligands with optimized selectivity.

• Dokumententyp: Zeitschriftenartikel
• Publikationsform: Publisher's Version
• Keywords: E. coli; recombinant expression; 4/7 α-conotoxin; ribbon isomer; molecular modeling; NMR spectroscopy
• Fakultät: Medizin
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• ISSN: 1663-9812
• Sprache: Englisch