Distinct Disulfide Isomers of μ-Conotoxins KIIIA and KIIIB Block Voltage-Gated Sodium Channels

Abstract

In the preparation of synthetic conotoxins containing multiple disulfide bonds, oxidative folding can produce numerous permutations of disulfide bond connectivities. Establishing the native disulfide connectivities thus presents a significant challenge when the venom-derived peptide is not available, as is increasingly the case when conotoxins are identified from cDNA sequences. Here, we investigate the disulfide connectivity of μ-conotoxin KIIIA, which was predicted originally to have a [C1-C9,C2-C15,C4-C16] disulfide pattern based on homology with closely-related μ-conotoxins. The two major isomers of synthetic μ-KIIIA formed during oxidative folding were purified and their disulfide connectivities mapped by direct mass spectrometric CID fragmentation of the disulfide-bonded polypeptides. Our results show that the major oxidative folding product adopts a [C1-C15,C2-C9,C4-C16] disulfide connectivity, while the minor product adopts a [C1-C16,C2-C9,C4-C15] connectivity. Both of these peptides were potent blockers of NaV1.2 (Kd 5 and 230 nM, respectively). The solution structure for μ-KIIIA based on NMR data was recalculated with the [C1-C15,C2-C9,C4-C16] disulfide pattern; its structure was very similar to the μ-KIIIA structure calculated with the incorrect [C1-C9,C2-C15,C4-C16] disulfide pattern, with an α-helix spanning residues 7–12. In addition, the major folding isomers of μ-KIIIB, an N-terminally extended isoform of μ-KIIIA identified from its cDNA sequence, were isolated. These folding products had the same disulfide connectivities as for μ-KIIIA, and both blocked NaV1.2 (Kd 470 and 26 nM, respectively). Our results establish that the preferred disulfide pattern of synthetic μ-KIIIA/μ-KIIIB folded in vitro is 1-5/2-4/3-6 but that other disulfide isomers are also potent sodium channel blockers. These findings raise questions about the disulfide pattern(s) of μ-KIIIA in the venom of Conus kinoshitai; indeed, the presence of multiple disulfide isomers in the venom could provide a means to further expand the snail's repertoire of active peptides.

Mammalian Neuronal Sodium Channel Blocker μ-Conotoxin BuIIIB Has a Structured N-Terminus That Influences Potency

“…In this study we have verified that the 1-4/2-5/3-6 pattern is correct for the major product of in vitro oxidative refolding. In contrast, recent studies on μ-KIIIA (37, 38) showed that the thermodynamically favored product of oxidative folding in vitro has 1-5/2-4/3-6 disulfide connectivity. We note that μ-KIIIA has a shorter first loop compared with other M5 μ-conotoxins, with only one amino acid residue between the second and third cysteine residues (Figure 1), and this is likely to influence folding and thus disulfide bridge formation.…”

“…* for μ-KIIIA indicates that the disulfide pattern differs from that determined here for μ-BuIIIB, with the major product from oxidative refolding in vitro adopting a 1-5/2-4/3-6 pattern rather than the 1-4/2-5/3-6 pattern for μ-BuIIIB. (38) ** for μ-SIIIA and μ-SmIIIA indicates that the disulfide patterns for the major products from oxidative refolding in vitro have not been determined experimentally as yet. (B) Amino acid sequences of μ-BuIIIB and [ d -Ala2]BuIIIB for this study.…”

Scrambling of disulfide bond scaffolds in neurotoxin AuIB: A molecular dynamics simulation study

“…11 For this purpose it is very important to know how crucial a particular disulfide bond is toward maintaining the structure and activity of the peptide. 9,10 Usually, it's the native form of the peptide, which is the most efficient, in terms of functional activity. However, there may be exceptions, e.g., in AuIB, it's the nonnative ribbon isoform (Scheme 1), which shows the highest potency.…”

“…The structural folds, on the other hand, are significantly responsible for regulating the toxin function and selectivity. [9][10][11] In general, peptides containing multiple disulfide linkages show strong dependence of their structural and functional discrimination on the mode of connectivity between the cysteine residues. Disulfide bond formation is a post-translational process, which helps fold a protein/peptide structure by restraining the distance and angle between the C b -S c atoms of the joined cysteine residues.…”

“…Prior experimental works and computer simulations have already pointed out the crucial involvement of multiple disulfide linkages in maintaining a peptide structure and function. 9,10,24,51 In a seminal work, Zhang and Snyder first showed the systematic dependence of a conopeptide structure on a particular disulfide bond. They extensively studied the wild type and several other synthetic variants of a-Conotoxin, GI.…”