Venomous predatory animals, such as snakes, spiders, scorpions, sea anemones, and
conesnails, produce a variety of highly stable cystine-
constrained peptide scaffolds as part of their neurochemicalstrategy for capturing prey. Here we report a new family of four-cystine, three-loop conotoxins (designatedframework 14). Three peptides of this family (flf14a-c) were isolated from the venom of
Conus floridanusfloridensis, and one (vil14a) was isolated from the venom of
Conus villepinii, two worm-hunting WesternAtlantic
cone snail species. The primary structure for these peptides was determined using Edmandegradation sequencing, and their cystine pairing was assessed by limited hydrolysis with a combinationof CNBr and chymotrypsin under nonreducing, nonalkylating conditions in combination with MALDI-TOF MS analysis of the resulting peptidic fragments. CD spectra and nanoNMR spectroscopy of theseconotoxins directly isolated from the
cone snails revealed a highly helical secondary structure for the fourconotoxins. Sequence-specific nanoNMR analysis at room temperature revealed a well-defined helix-loop-helix tertiary structure that resembles that of the Cs
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scorpion toxins
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-hefutoxin,
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-KTx1.3,and Om-toxins, which adopt a stable three-dimensional fold where the two
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-helices are linked by thetwo disulfide bridges. One of these conotoxins (vil14a) has a Lys/Tyr dyad, separated by approximately6Å, which is a conserved structural feature in K
+ channel blockers. The presence of this framework inscorpions and in
cone snails indicates a common molecular imprint in the venom of apparently unrelatedpredatory animals and suggests a common ancestral genetic origin.