Definition of the R-superfamily of conotoxins: Structural convergence of helix-loop-helix peptidic scaffolds

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HighlightsUsing the transcriptome and cDNA libraries, we determined the precursor proteins sequences that define the R-superfamily of conotoxins.The R-superfamily pre-pro region is unusually long and with prevalent Pro repeats, which constitutes a proline-rich motif (PRM).We determined the three dimensional structure of vil14a, which displays a cystine-stabilized α-helix-loop-helix (Cs α/α) fold.The R-superfamily conotoxins overlap with Cs α/α scorpion toxins and other native peptides indicating structural convergence for this motif.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 mélange. 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 Å), 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.

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