Original Research ARTICLE
Periplasmic expression of 4/7 α-conotoxin TxIA analogues in E. coli favours ribbon isomer formation - Suggestion of a binding mode at the α7 nAChR
- 1Walther Straub Institute of Pharmacology and Toxicology, Faculty of Medicine, Ludwig Maximilians University Munich, Germany
- 2Institute for Molecular Bioscience, University of Queensland, Australia
- 3School of Biomedical Sciences, University of Queensland, Australia
- 4UMR5247 Institut des Biomolécules Max Mousseron (IBMM), France
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 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 α-conotoxin TxIA, a potent blocker at α7 and α3β2 nicotinic acetylcholine receptors (nAChRs), and three analogues 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 analogues confirmed that the decreased activity was due to incorrect disulfide linkage rather than the missing C-terminal amidation, a posttranslational modification that is commonl in α-conotoxins. All peptides preferentially formed in the ribbon conformation rather than the native globular conformation. Interestingly, in the case of the α7 nAChR, but not the α3β2 subtype, the loss of potency could be rescued by an R5D substitution. In conclusion, we demonstrate efficient expression of functional but incorrectly folded ribbon TxIA variants in E. coli and provide the first structure-function analysis for a ribbon 4/7-α-conotoxin at α7 and α3β2 nAChRs. Computational analysis based on these data provide evidence for a ribbon α-conotoxin binding mode that might be exploited to design ligands with optimized selectivity.
Keywords: E. coli, Recombinant expression, 4/7 α-conotoxin , ribbon isomer , Molecular modeling
Received: 08 Feb 2019;
Accepted: 06 May 2019.
Edited by:Yasuo Mori, Kyoto University, Japan
Reviewed by:Oscar Moran, Istituto di biofisica (IBF), Italy
Shigeki Kiyonaka, Kyoto University, Japan
Copyright: © 2019 El Hamdaoui, Wu, Clark, Giribaldi, Anangi, Craik, King, Dutertre, Kaas, Herzig and Nicke. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Dr. Quentin Kaas, Institute for Molecular Bioscience, University of Queensland, St Lucia, 4072, Queensland, Australia, email@example.com
Dr. Volker Herzig, Institute for Molecular Bioscience, University of Queensland, St Lucia, 4072, Queensland, Australia, firstname.lastname@example.org
Prof. Annette Nicke, Walther Straub Institute of Pharmacology and Toxicology, Faculty of Medicine, Ludwig Maximilians University Munich, Munich, Bavaria, Germany, email@example.com