Structural Characterization and Biological Potency of Novel Conotoxin MIIIB from Conus Magus

Date
2020
Authors
Zhang, Rui-Yang
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Bingham, Jon-Paul
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Molecular Biosciences and Bioengineering
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Abstract
Cone snail venoms, in particular, the peptide toxins or so-called conotoxins, provide an untapped resource for neuropharmacological tools and therapeutics candidates. Thus, conotoxins have turned into a research hotspot in neuroscience and new drug development during the past decades. Although cone snails generate the variety and abundance nature of conotoxins, less than 0.1% of the estimated conotoxins have been characterized and documented to date. Therefore, the discovery of novel conotoxins from the venom repertoire combined with their structural traits and biological activity characterization becomes crucial for the conotoxin-based therapeutic development. Here, we present the structural and biological activity characterization of a novel Mini-M conotoxin, MIIIB, which was isolated from the reduced milked venom pool of Conus magus. The characterization of this novel peptide-toxin was achieved through peptide synthesis, oxidation profiling, LD50 bioassay, disulfide patterns elucidation, mutant productions, and NMR structure determination. From the analyses, we revealed that (1) MIIIB is capable of forming six dominant disulfide isomers (A-F), and the isomer F not only demonstrates the most significant LD50 value as 1.75 µg/g but considered as the native-like conformation of MIIIB. (2) the disulfide connectivity of the MIIIB isomer F is determined as [C1-C6, C2-C4, C3-C5], which is the first case demonstrating the alternative disulfide connectivity exists in an M-superfamily conotoxin branch. (3) the unusual N-terminal Met is essential in MIIIB folding, also may play a critical role in the bioactivity in regards to the ligand-receptor binding event. (4) the soluble structure of MIIIB isomer F indicates that its unique conformation is distinct from any known Mini-M conotoxins but structurally analogous to χ-conotoxin MrIA and Poneritoxin Ae1a, though different cysteine frameworks and disulfide patterns reside in these peptides. (5) MIIIB incorporates inter-cysteine loop size as 5-9-1, and this extended loop might be the evolutionary consequence of feeding strategy swapping within cone snails; thus, 5-9-1 loop size might only be seen in fish-eating cone snails. These findings implicate how the amino acid sequence diversity influences the disulfide formation and structural conformation. Herein, we believe the MIIIB isomer F provides a new scaffold for peptide drug design based on the biological potency and a future target for peptide bioengineering to enhance receptor-targeting selectivity and specificity.
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Biochemistry, Conotoxins, Disulfide Connectivity, M-superfamily, N-terminal Methionine, Nuclear Magnetic Resonance, Peptides
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206 pages
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