title: 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 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 μ-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 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 μ-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 μ-KIIIA and 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 of further expanding the snail's repertoire of active peptides.
authors.affiliation: Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, Australia.
authors.forename: Keith K
authors.initials: KK
authors.lastname: Khoo
authors.forename: Kallol
authors.initials: K
authors.lastname: Gupta
authors.forename: Brad R
authors.initials: BR
authors.lastname: Green
authors.forename: Min-Min
authors.initials: MM
authors.lastname: Zhang
authors.forename: Maren
authors.initials: M
authors.lastname: Watkins
authors.forename: Baldomero M
authors.initials: BM
authors.lastname: Olivera
authors.forename: Padmanabhan
authors.initials: P
authors.lastname: Balaram
authors.forename: Doju
authors.initials: D
authors.lastname: Yoshikami
authors.forename: Grzegorz
authors.initials: G
authors.lastname: Bulaj
authors.forename: Raymond S
authors.initials: RS
authors.lastname: Norton
mesh_terms: D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D020916:Conotoxins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D004220:Disulfides / Q000737:chemistry*; D015640:Ion Channel Gating; D007536:Isomerism; D013058:Mass Spectrometry; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D015222:Sodium Channels / Q000187:drug effects*
chemical_list: D020916:Conotoxins; D004220:Disulfides; D015222:Sodium Channels