The structure of spider toxin huwentoxin-II with unique disulfide linkage: evidence for structural evolution

Abstract

The three-dimensional structure of huwentoxin-II (HWTX-II), an insecticidal peptide purified from the venom of spider Selenocosmia huwena with a unique disulfide bond linkage as I-III, II-V, and IV-VI, has been determined using 2D (1)H-NMR. The resulting structure of HWTX-II contains two beta-turns (C4-S7 and K24-W27) and a double-stranded antiparallel beta-sheet (W27-C29 and C34-K36). Although the C-terminal double-stranded beta-sheet cross-linked by two disulfide bonds (II-V and IV-VI in HWTX-II, II-V and III-VI in the ICK molecules) is conserved both in HWTX-II and the ICK molecules, the structure of HWTX-II is unexpected absence of the cystine knot because of its unique disulfide linkage. It suggests that HWTX-II adopts a novel scaffold different from the ICK motif that is adopted by all other spider toxin structures elucidated thus far. Furthermore, the structure of HWTX-II, which conforms to the disulfide-directed beta-hairpin (DDH) motif, not only supports the hypothesis that the ICK is a minor elaboration of the more ancestral DDH motif but also suggests that HWTX-II may have evolved from the same structural ancestor.

Fig. 1.

Comparison of disulfide bond linkage pattern of huwentoxin-II, huwentoxin-I, ω-agatoxin IVA, δ-atracotoxin-Hv1b, and J-atracotoxin Hv1c. Huwentoxin-II has a unique disulfide-pairing mode as I-III, II-V, and IV-VI. Huwentoxin-I, ω-agatoxin IVA, δ-atracotoxin-Hv1b, and J-atracotoxin Hv1c represent four kinds of disulfide pairing modes found in spider toxins that adopt the inhibitor cystine knot motif in three-dimensional structures.

Fig. 2.

Three-dimensional solution structure of HWTX-II. (A) The backbone atom superimposition plot for the ensemble of 10 HWTX-II structures. The side chain heavy atoms of the disulfide bonds are shown. (B) The secondary topology of HWTX-II. The double-stranded antiparallel β-sheet is shown in yellow, and turns in blue, random coil structure is shown in green. The three disulfide bonds (C4-C18, C8-C29, and C23-C34) are indicated. The three-dimensional structure of HWTX-II consists of the N- and C-terminal parts and two connecting elements (the fragment 19–22 and the disulfide C8-C29). (C) The charged residues in HWTX-II form a dipolar distribution, and the side chains of residue W27, M33, and V35 distributed around the disulfides C8-C29 and C23-C34 form hydrophobic patches. Both in A and C, the colors of the C, N, O, and S atom are green, blue, red, and yellow, respectively.

Fig. 3.

Comparison between HWTX-II (A) and the inhibitor cystine knot motif molecules (B–E) with different disulfide bond numbers and linkage patterns. The backbone folding (in green), the sheet structures (in yellow) classified by Kabsch-Sander method in InsightII, and disulfide bonds are shown. (A) HWTX-II. Three disulfide bonds link in I-III, II-V, and IV-VI (C4-C18, C8-C29, and C23-C34) mode. (B) Huwentoxin-I. Three disulfides shown in yellow are linked in the typical ICK motif mode as I-IV, II-V, and III-VI (C2-C17, C9-C22, and C16-C29) (cf. Fig. 1 ▶). The III-VI disulfide passes through a ring formed by the I-IV and II-V disulfide bonds and the intervening peptide backbone, it is known as the cystine knot. (C) δ-atracotoxin-Hv1b. Four disulfides linked in C1-C15, C8-C20, C14-C31, and C16-C42 (cf. Fig. 1 ▶). (D) ω-agatoxin IVA. Four disulfides linked in C4-C20, C12-C25, C19-C36, and C27-C34 (cf. Fig. 1 ▶). (E) J-atracotoxin Hv1c. Four disulfides linked in C3-C17, C10-C22, C13-C14, and C16-C33 (cf. Fig. 1 ▶). The disulfide bonds involving in the cystine knot (I-IV, II-V, and III-VI) in the ICK motif structures, i.e. B, C, D, and E, are shown in yellow. The fourth additional disulfides (shown in red in B–E) in the ICK folding molecules are various and not involved in the cystine knot such as C16-C42 in δ-atracotoxin-Hv1b (C), C27-C34 in ω-agatoxin IVA (D), and C13-C14 in J-atracotoxin Hv1c (E). The ICK motif spider toxins with different disulfide bond numbers and linkages (B–E) all form the cystine knot, whereas HWTX-II (A) does not because of its unique disulfide bridges linkage. Moreover, the II-V and the IV-VI disulfide bond of HWTX-II (shown in yellow in A) dictating the C-terminal β-hairpin are similar to corresponding ones in ICK folding molecules (B–E). The disulfide I-III of HWTX-II (shown in red in A) is different from the corresponding ones of ICK folding molecules.

Fig. 4.

Sequence alignment of HWTX-II, TX4K EURCA from Eurypelma californicum and TXP1 BRASM from Brachypelma smithii. TX4K EURCA and TXP1 BRASM are the ID numbers of Swissprot database to ESTX and venom protein 1, respectively. Gaps have been inserted to achieve the best alignment. Residues conserved for HWTX-II, ESTX, and venom protein 1 are shaded and residues identical among the three molecules are given against a black background.

Fig. 5.

Comparison of the structure of J-atracotoxin Hv1c, the CBD-CBHI, and HWTX-II. (A) J-atracotoxin Hv1c; (B) CBD-CBHI; (C) HWTX-II. The double-stranded β-sheet dictated by the two cross-linked disulfides in the C-terminal part (shown in yellow) is conserved in three structures.