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The F14 conotoxins define a four-cysteine, three-loop conotoxin scaffold that produce tightly folded structures held together by two disulfide bonds with a C C C C arrangement (conotoxin framework 14). Here we describe the precursors of the F14 conotoxins from the venom of Conus anabathrum and Conus villepinii. Using transcriptomic and cDNA cloning analysis, the full-length of the precursors of flf14a and flf14b from the transcriptome of C. anabathrum revealed a unique signal sequence that defines the new conotoxin R-superfamily. Using the signal sequence as a primer, we cloned seven additional previously undescribed toxins of the R-superfamily from C. villepinii. The propeptide regions of the R-conotoxins are unusually long and with prevalent proline residues in repeating pentads which qualifies them as Pro-rich motifs (PRMs), which can be critical for protein-protein interactions or they can be cleaved to release short linear peptides that may be part of the envenomation melange. Additionally, we determined the three-dimensional structure of vil14a by solution 1H-NMR and found that the structure of this conotoxin displays a cysteine-stabilized α-helix-loop-helix (Cs α/α) fold. The structure is well-defined over the helical regions (backbone RMSD for residues 2–13 and 17–26 is 0.63 ± 0.14 A), with conformational flexibility in the triple Gly region of the second loop as well as the N- and C- termini. Structurally, the F14 conotoxins overlap with the Cs α/α scorpion toxins and other peptidic natural products, and in spite of their different exogenomic origins, there is convergence into this scaffold from several classes of living organisms that express these peptides.

Venoms from cone snails (genus Conus) can be seen as an untapped cocktail of biologically active compounds, being increasingly recognized as an emerging source of peptide-based therapeutics. Cone snails are considered to be specialized predators that have evolved the most sophisticated peptide chemistry and neuropharmacology system for their own biological purposes by producing venoms which contains a structural and functional diversity of neurotoxins. These neurotoxins or conotoxins are often small cysteine-rich peptides which have shown to be highly selective ligands for a wide range of ion channels and receptors. Local habitat conditions have constituted barriers preventing the spreading of Conus species occurring along the coast of South Africa. Due to their scarceness, these species remain, therefore, extremely poorly studied. In this work, the venoms of two South African cone snails, Conus pictus, a vermivorous snail and Conus natalis, a molluscivorous snail, have been characterized in depth. In total, 26 novel peptides were identified. Comparing the venoms of both snails, interesting differences were observed regarding venom composition and molecular characteristics of these components.