Sequence-specific 1H-NMR assignment and determination of the secondary structure of hainantoxin-III from the spider Ornithoctonus hainana.

Hainantoxin-III (HNTX-III) purified from the venom of the spider Ornithoctonus hainana is a novel neurotoxin preferentially inhibiting tetrodotoxin-sensitive voltage-gated sodium channels in rat dorsal root ganglion cells. The structure of this toxin in aqueous solution was investigated using 2-D H-1-NMR techniques. The complete sequence-specific assignments of proton resonances in the H-1-NMR spectra were obtained by analyzing a series of 2-D spectra, including DQF-COSY, TOCSY and NOESY spectra, in H2O or D2O. All the backbone protons and more than 95% of the side-chain protons have been assigned by d(alpha N), d(beta N), and d(NN) connectivities in NOESY spectrum. Furthermore, the secondary structure of HNTX-III was identified from NMR data. It consists mainly of a short triple-stranded antiparallel beta-sheet formed by Asp7 to Cys9, Tyr21 to Ser23, and Lys27 to Val30. Because HNTX-III shares high sequence identity (>70%) with HWTX-I and HNTX-I, we proposed that they all share a structural scaffold known as the inhibitor cystine knot architectural motif. This study provides a basis for the further determination of the solution conformation of HNTX-III.