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.

Figure 1

Representative reversed-phase HPLC chromatograms of the oxidative folding of (A) μ-KIIIA and (B) μ-KIIIB. μ-KIIIA Peaks 1 and 2 represent the [C1-C15,C2-C9,C4-C16] and [C1-C16,C2-C9,C4-C15] connectivities, respectively (see text). Folding was carried out in buffered solution (pH 7.5) containing a 1:1 mM mixture of oxidized and reduced glutathione for 2 h at room temperature.

Figure 2

CID MS² spectrum of the peptide derived upon trypsin digestion of μ-KIIIA-P1 (712.8, (M+2H)²⁺). Inset shows the MS spectrum of the precursor ion and MS³ spectra of 995.2.

Figure 3

Assignments of the key MSⁿ fragment ions of tryptic μ-KIIIA-P1. The m/z values of each of the ions are indicated against the respective structures and they correspond to the singly charged values, unless otherwise specified. For every structure, Cys residues with indeterminate connectivity are indicated with the wavy lines. Subsequently, in the structures from which a particular Cys connectivity is evident, the connected Cys residues are joined through a dashed line. The arrows indicate the site of proteolysis. The 2–4 connectivity is established by the product ion 488.2, while the ions 656.2 and 791.2 confirm the 3–6 connectivity.

Figure 4

CID MS² spectrum of the peptide derived upon trypsin digestion of μ-KIIIA-P2 (712.8, (M+2H)²⁺). Inset shows the extracted ion chromatogram from the LC-MS analysis of μ-KIIIA-P1, P2 and a reduced equimolar mixture of both fractions.

Figure 5

Stereo views of backbone and closest-to-average structures of μ-KIIIA[C1-C15,C2-C9,C4-C16] (green) and μ-KIIIA[C1-C9,C2-C15,C4-C16] (blue). Disulfide bonds are displayed in yellow.

Figure 6

Stereo views of overlay of ensemble of 20 NMR structures of μ-KIIIA[C1-C15,C2-C9,C4-C16] (green) and μ-KIIIA[C1-C9,C2-C15,C4-C16] (blue), superimposed over backbone heavy atoms of residues 4–16. Top and bottom panel views are related by a 90° anticlockwise rotation about the vertical-axis.