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

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.

α-Conotoxin as Potential to α7-nAChR Recombinant Expressed in Escherichia coli

“…To date, over 11 conotoxins (α-TxIA, TxIB, LvIA, M-superfamily lt16a, lt15a, Bt15a, Ec15a, Cap15a, Vx15a, Vr15a, lt7a, μO-MrVIB and ω-MVIIa, PrIIIE, etc.) have successfully been recombinantly expressed in Escherichia coli [ 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 ] and bioactive TxVIA has been produced in the yeast Pichia pastoris [ 43 ].…”

Structure-Function Elucidation of a New α-Conotoxin, MilIA, from Conus milneedwardsi

“…Among the α3/5 conotoxins, α-conotoxins MI, GI, and SI are the three most investigated peptides. The IC 50 values of these conotoxins range from 20–40 nM, with an exception for α-conotoxin MI with an IC 50 value of 0.4 nM [20]. Remarkably different from this data, was that native α-conotoxin MilIA showed a low potency, towards the muscle type nAChR (IC 50 fetal = 13,130 ± 1125 nM and IC 50 adult = 1118 ± 78,891 nM).…”

An environmentally sustainable biomimetic production of cyclic disulfide-rich peptides

“…Although native producers of disulfide-rich peptides tend to generate only a single native isomer that has optimal activity, heterologous production in a different organism may produce non-native isomers. 51 To verify correct folding of the P. pastoris-expressed peptides, we focused on [G22N]cVc1.1 and MCoTI-II as examples of peptides with two and three disulfide bonds, respectively (SFTI-based peptides have only one disulfide bond).…”

“…32 Several studies have attempted to produce -conotoxins recombinantly but these previous reports either did not perform structural studies or failed to produce the native "globular" isomer. 51,58,59 Using our production platform, we demonstrated the expression of [G22N]cVc1.1 in the native globular conformation. It is known that non-native -conotoxin isomers can lose potency and/or specificity.…”