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 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 α3β2 and α7 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 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 alternatively 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.
Highlights
Nicotinic acetylcholine receptors are members of the Cys-loop superfamily of pentameric ligand-gated ion channels
The DNA sequences corresponding to TxIA and analogs were optimized for expression in E. coli and fused N-terminally via a tobacco etch virus (TEV) protease cleavage site to maltose binding protein (MBP) (Figure 1B)
The consensus TEV protease cleavage site is ENLYFQ/S, the protease efficiently cleaves at ENLYFQ/G (Kapust et al, 2002), which has the advantage that the N-terminal glycine residue that is present in most α-conotoxins can be used and remains after cleavage
Summary
Nicotinic acetylcholine receptors (nAChRs) are members of the Cys-loop superfamily of pentameric ligand-gated ion channels. Whereas the muscle-type receptors consist of four different subunits (α1,β1,γ/ε,δ), the assembly and stoichiometry of the 11 cloned neuronal subunits (α2–α7, α9, α10, and β2–β4) into nAChR subtypes is considerably more diverse and not welldefined These neuronal nAChRs represent important drug targets such as for the treatment of pain, Alzheimer’s disease, and nicotine addiction (Dineley et al, 2015; Lombardo and Maskos, 2015; Mohamed et al, 2015; Giribaldi and Dutertre, 2018; Hone and McIntosh, 2018). The best-studied conotoxin family from a pharmacological perspective is the α-conotoxins, members of which are competitive antagonists of nAChRs (Dutertre et al, 2017) Some of these peptides have analgesic activity in vivo and are important lead structures for drug development (Akondi et al, 2014; Mohammadi and Christie, 2015)
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