Proline hydroxylation and C-terminal amidation in µ-conotoxins increase structural stability and potency at sodium channels

Victoria A. Adegoke; Yashad Dongol; Tye Gonzalez; Angela Song; Richard J. Clark; Richard J. Lewis; Anne C. Conibear; K. Johan Rosengren

doi:10.1071/CH25071

RESEARCH ARTICLE (Open Access)

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Proline hydroxylation and C-terminal amidation in µ-conotoxins increase structural stability and potency at sodium channels

Victoria A. Adegoke ^A , Yashad Dongol ^B , Tye Gonzalez ^A , Angela Song ^A , Richard J. Clark ^A , Richard J. Lewis

^B , Anne C. Conibear

^A ^C ^* and K. Johan Rosengren ^A ^*

+ Author Affiliations

- Author Affiliations

^A School of Biomedical Sciences, Faculty of Medicine, The University of Queensland, Saint Lucia, Qld 4072, Australia.

^B Institute for Molecular Bioscience, The University of Queensland, Saint Lucia, Qld 4072, Australia.

^C Institute of Applied Synthetic Chemistry, Faculty of Technical Chemistry, TU Wien, Getreidemarkt 9, A-1060 Vienna, Austria.

^* Correspondence to: anne.conibear@tuwien.ac.at, j.rosengren@uq.edu.au

Handling Editor: Mibel Aguilar

Australian Journal of Chemistry 78, CH25071 https://doi.org/10.1071/CH25071

Submitted: 30 April 2025 Accepted: 22 July 2025 Published online: 2 September 2025

© 2025 The Author(s) (or their employer(s)). Published by CSIRO Publishing. This is an open access article distributed under the Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC)

Abstract

Conotoxins are disulfide-rich peptides isolated from the venoms of marine cone snails. These natural products have inspired the development of several drug candidates and novel therapeutic leads. In addition to disulfide bonds, many conotoxins are highly modified with posttranslational modifications (PTMs) such as proline hydroxylation, C-terminal amidation and glycosylation, among others. These modifications can alter the charge, size and hydrophobicity of the conotoxin, influencing its interaction with target receptors and modulating its potency and selectivity. PTMs can also affect the folding kinetics and conformational stability of the peptide, which further affects its biological activity. Although conotoxins undergo a variety of PTMs, the functions of many of these modifications remain unclear. Here, we explored the structural and functional implications of PTMs in two representative conotoxins, PIIIA and TIIIA of the µ-pharmacological family. We synthesised a series of PIIIA and TIIIA peptides bearing native hydroxyproline and C-terminal amidation PTMs, along with their unmodified counterparts. Solid phase peptide synthesis and non-selective disulfide bond formation provided access to pure forms of the eight possible variants for in vitro comparison of their oxidative folding. Structural studies using nuclear magnetic resonance (NMR) spectroscopy, alongside electrophysiological and serum stability assays, were conducted to characterise the functional roles of the PTMs in these conotoxins. Our results suggest that, whereas C-terminal amidation has a crucial role in folding and structural integrity, proline hydroxylation significantly influences the in vitro oxidative folding, stability and biological activity of these conotoxin peptides.

Keywords: C-terminal amidation, µ-conotoxins, nuclear magnetic resonance spectroscopy, oxidative folding, posttranslational modification, proline hydroxylation, sodium channels, solid phase peptide synthesis, structure–function relationship.

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