2LXG

NMR solution structure of Mu-conotoxin KIIIA

Summary for 2LXG

• Entry DOI: 10.2210/pdb2lxg/pdb
• NMR Information: BMRB: 20048
• Descriptor: Mu-conotoxin KIIIA (1 entity in total)
• Functional Keywords: conotoxin, disulfide isomer, toxin, voltage gated sodium channels, helical
• Biological source: Conus kinoshitai (Kinoshita's cone)
• Cellular location: Secreted (By similarity): P0C195
• Total number of polymer chains: 1
• Total formula weight: 1893.23

Authors

Khoo, K.K.,Norton, R.S. (deposition date: 2012-08-21, release date: 2013-02-06, Last modification date: 2024-10-30)

Primary citation

Khoo, K.K.,Gupta, K.,Green, B.R.,Zhang, M.M.,Watkins, M.,Olivera, B.M.,Balaram, P.,Yoshikami, D.,Bulaj, G.,Norton, R.S.
Distinct disulfide isomers of mu-conotoxins KIIIA and KIIIB block voltage-gated sodium channels.
Biochemistry, 51:9826-9835, 2012

PubMed 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.

PubMed: 23167564
DOI: 10.1021/bi301256s

Experimental method

SOLUTION NMR