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

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• 作者：Keith K. Khoo ; Kallol Gupta ; Brad R. Green ; Min-Min Zhang ; Maren Watkins ; Baldomero M. Olivera ; Padmanabhan Balaram ; Doju Yoshikami ; Grzegorz Bulaj ; Raymond S. Norton
• 刊名：Biochemistry
• 出版年：2012
• 出版时间：December 11, 2012
• 年：2012
• 卷：51
• 期：49
• 页码：9826-9835
• 全文大小：429K
• 年卷期：v.51,no.49(December 11, 2012)
• ISSN：1520-4995

文摘

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 渭-conotoxin KIIIA, which was predicted originally to have a [C1鈥揅9,C2鈥揅15,C4鈥揅16] disulfide pattern based on homology with closely related 渭-conotoxins. The two major isomers of synthetic 渭-KIIIA formed during oxidative folding were purified and their disulfide connectivities mapped by direct mass spectrometric collision-induced dissociation fragmentation of the disulfide-bonded polypeptides. Our results show that the major oxidative folding product adopts a [C1鈥揅15,C2鈥揅9,C4鈥揅16] disulfide connectivity, while the minor product adopts a [C1鈥揅16,C2鈥揅9,C4鈥揅15] connectivity. Both of these peptides were potent blockers of Na_V1.2 (K_d values of 5 and 230 nM, respectively). The solution structure for 渭-KIIIA based on nuclear magnetic resonance data was recalculated with the [C1鈥揅15,C2鈥揅9,C4鈥揅16] disulfide pattern; its structure was very similar to the 渭-KIIIA structure calculated with the incorrect [C1鈥揅9,C2鈥揅15,C4鈥揅16] disulfide pattern, with an 伪-helix spanning residues 7鈥?2. In addition, the major folding isomers of 渭-KIIIB, an N-terminally extended isoform of 渭-KIIIA identified from its cDNA sequence, were isolated. These folding products had the same disulfide connectivities as 渭-KIIIA, and both blocked Na_V1.2 (K_d values of 470 and 26 nM, respectively). Our results establish that the preferred disulfide pattern of synthetic 渭-KIIIA and 渭-KIIIB folded in vitro is 1鈥?/2鈥?/3鈥? but that other disulfide isomers are also potent sodium channel blockers. These findings raise questions about the disulfide pattern(s) of 渭-KIIIA in the venom of Conus kinoshitai; indeed, the presence of multiple disulfide isomers in the venom could provide a means of further expanding the snail鈥檚 repertoire of active peptides.