Redox-Active Conopeptide Li520 Has Evolved to Catalyze Oxidative Folding of Conotoxins

The evolution of miniature conopeptide Li520 (COWC*,
*: C-terminal
amidation) to exhibit the disulfide isomerase activity was probed
using structure, function, disulfide conformation, and the precursor
gene sequence. The peptides Li520, Li504, [O2A]Li520, [W3A]Li520,
and Grx506, homologues active-site motif of glutaredoxin, were chemically
synthesized and assessed for their disulfide reduction potential,
intrinsic folding of disulfides, and disulfide isomerization activity
on α-conotoxin ImI. The reduction potential of the disulfide
of peptides varies from −189 to −344 mV, which is within
the range observed for the redox family of proteins that modulates
the folding of protein disulfides. The oxidative folding studies confirm
the significance of the tryptophan residue in engaging Li520 in disulfide-exchange
reactions and the role of proline hydroxylation in extending the lifetime
of Li520 in a reduced free thiol state. Studies of quenching of tryptophan
fluorescence by the disulfide in situ folding reaction in conjunction
with the optimized structures by density functional theory (DFT) confirm
the difference in conformation of disulfides between the native and
mutant peptides. Interestingly, the native peptide Li520/Li504 shares
a similar disulfide conformation of (−,−)AntiRHHook
with the redox family of proteins known to modulate disulfides, particularly
in lieu of the tetrapeptide of glutaredoxin, deviating from its disulfide
conformation compared to its naive protein. Analysis of the precursor
gene sequences of M-superfamily conotoxins revealed the presence of
Li520 in different cone snail species with distinct food habits and
possible modes of evolution through the diversification of cysteine
motifs. The results of the report suggest that the short redox conopeptide
Li520 has evolved to facilitate the oxidative folding of conotoxins
and may be useful to develop as reagents for the synthesis of therapeutically
important cysteine-rich peptides.