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

Khoo, Keith K and Gupta, Kallol and Green, Brad R and Zhang, Min-Mm and Watkins, Maren and Olivera, Baldomero M and Balaram, Padmanabhan and Yoshikami, Doju and Bulaj, Grzegorz and Norton, Raymond S (2012) Distinct Disulfide Isomers of mu-Conotoxins KIIIA and KIIIB Block Voltage-Gated Sodium Channels. In: BIOCHEMISTRY, 51 (49). pp. 9826-9835.

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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 mu-conotoxin KIIIA, which was predicted originally to have a C1-C9,C2-C15,C4-C16] disulfide pattern based on homology with closely related mu-conotoxins. The two major isomers of synthetic mu-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-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 Na(v)1.2 (K-d values of 5 and 230 nM, respectively). The solution structure for mu-KIIIA based on nuclear magnetic resonance data was recalculated with the C1-C15,C2-C9,C4-C16] disulfide pattern; its structure was very similar to the mu-KIIIA structure calculated with the incorrect C1-C9,C2-C15,C4-C16] disulfide pattern, with an alpha-helix spanning residues 7-12. In addition, the major folding isomers of mu-KIIIB, an N-terminally extended isoform of mu-KIIIA, identified from its cDNA sequence, were isolated. These folding products had the same disulfide connectivities as mu-KIIIA, and both blocked Na(v)1.2 (K-d values of 470 and 26 nM, respectively). Our results establish that the preferred disulfide pattern of synthetic mu-KIIIA and mu-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 mu-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's repertoire of active peptides.

Item Type:	Journal Article
Publication:	BIOCHEMISTRY
Publisher:	AMER CHEMICAL SOC
Additional Information:	Copyright for this article belongs to AMER CHEMICAL SOC, WASHINGTON
Department/Centre:	Division of Biological Sciences > Molecular Biophysics Unit
Date Deposited:	16 Jan 2013 12:42
Last Modified:	16 Jan 2013 12:42
URI:	http://eprints.iisc.ac.in/id/eprint/45640

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