The Redox-Active Conopeptide Derived from the Venom Duct Transcriptome of Conus lividus Assists in the Oxidative Folding of Conotoxin

Abstract

The tetrapeptides Li504 and Li520, differing in the modification of the 4trans-hydroxylation of proline, are novel conopeptides derived from the venom duct transcriptome of the marine cone snail Conus lividus. These predicted mature peptides are homologous to the active site motif of oxidoreductases that catalyze the oxidation, reduction, and rearrangement of disulfide bonds in peptides and proteins. The estimated reduction potential of the disulfide of Li504 and Li520 is within the range of disulfide reduction potentials of oxidoreductases, indicating that they may catalyze the oxidative folding of conotoxins. Conformational features of Li504 and Li520 include the trans configuration of the Cys1−Pro2/Hyp2 peptide bond with a type 1 turn that is similar to the active site motif of glutaredoxin that regulates the oxidation of cysteine thiols to disulfides. Li504-and Li520-assisted oxidative folding of α-conotoxin ImI confirms that Li520 improves the yield of the natively folded peptide by concomitantly decreasing the yield of the non-native disulfide isomer and thus acts as a miniature disulfide isomerase. The geometry of the Cys1−Hyp2 peptide bond of Li520 shifts between the trans and cis configurations in the disulfide form and thiol/thiolate form, which regulates the deprotonation of the N-terminal cysteine residue. Hydrogen bonding of the hydroxyl group of 4-trans-hydroxyproline with the interpeptide chain unit in the mixed disulfide form may play a vital role in shifting the geometry of the Cys1−Hyp2 peptide bond from cis to trans configuration. The Li520 conopeptide together with similar peptides derived from other species may constitute a new family of "redox-active" conopeptides that are integral components of the oxidative folding machinery of conotoxins.

Oxidative Folding Catalysts of Conotoxins Derived from the Venom Duct Transcriptome of C. frigidus and C. amadis

“…identified a new class of folding catalysts of conotoxins called "redox-active conopeptides". 24 These conopeptides are homologous to the active site motifs of redox family of proteins 47−49 and assist in the formation of correct disulfide connectivities in conotoxins. Unlike PDIase, CsPDIase, and PPI, redox conopeptides were expressed similarly to conotoxins as precursor prepropeptides with the mature peptides located at the C-terminus.…”

“…The redox conopeptide-assisted oxidative folding of α-conotoxin ImI was carried out as described previously. 24 The purified reduced αconotoxin ImI (50 μM) was incubated with the reduced conopeptides Fr874, Fr890[P1O], Fr890[P2O], Fr906, Am1038, and Am1054 (5 mM) in the redox buffer of 1:1 GSH/GSSG (1 mM each) and 100 mM NH 4 HCO 3 (pH 8). The folding reaction was performed at room temperature and at pH 8 for about 1−3 h. The reaction was quenched using acidification with TFA at regular intervals of time.…”

“…11,12,14 Ashwini et al have demonstrated a new class of redox conopeptides that shares the active site motif of disulfide isomerases and catalyzes the formation of disulfide bonds of conotoxins. 24 Unlike other folding components of conotoxins such as PDI and peptidylprolyl cis−trans isomerase (PPI), 11,12,14,23 the redox conopeptides express as a precursor prepropeptide with a mature peptide located at the C-terminus, which has an advantage of generating multiple folding catalysts from the same sequence through post-translational modifications. Like the ω-conotoxin family of conotoxins 1,25,26 having distinct sequences with different affinities on the subunit of Ca 2+ channels, similar diversity may be expected in the sequences of the new family of redox conopeptides with distinct catalytical functions on oxidative folding of conotoxins.…”

“…The reversal of the configuration of tryptophan residues in contryphans 28 and post-translational modifications of proline residues in various conotoxins have been demonstrated to assist the oxidative folding of conotoxins. 24,29 The report evaluated the catalytic activity of new redox conopeptides using α-conotoxin ImI as a model peptide. Computational methods using density functional theory (DFT) and 2D-NMR methods have been implemented to probe the structural features of new redox conopeptides.…”

Marine natural products

“…Genome mining of C. lividus and subsequent peptide synthesis afforded two tetrapeptide mature peptides Li504 1468 and Li520 1469 , the sequence and molecular modelling of which identified similarity with the active site motif of disulfide-associated oxidoreductases. 649 The latter peptide was able to assist the oxidative folding of α-conotoxin ImI, affording improved yield of the natively folded protein.…”