Solution Structure Identification Of Huwentoxin-XVIIb1 By NMR

Evolution has equipped spider with a complex arsenal of neurotoxins to paralyze and kill their prey. Spider venoms are incredibly complex mixtures, comprising mainly protein and peptide toxins, which act on different ion channels or different subtype of ion channels. Due to their high affinity and specificity, a variety of peptide toxins of spider have been extensively used as structural and pharmacological tools for studying ion channels. Therefore, studies on identifying the structures of spider peptide toxins are significant to investigating the molecular mechanism and the relationship of the structure-function of ion channels.Chinese tarantula Selenocosmia huwena Wang [=Ornithoctonus huwena (Wang)], one of the largest and most venomous spiders of China, is distributed in Guangxi province. It is an attractive source of venom material due to its large size, the ability to breed in captivity and the availability of large amounts of venom. Huwentoxin-XVIIb1 (HWTX-XVIIb1) is a novel peptide neurotoxin, isolated and purified from the Chinese bird spider Selenocosmia huwena Wang. It contains 31 amino acid residues including 8 cysteines. Investigating on the biological function of this toxin, it was found that HWTX-XVIIb1 has no significant effect on Na+ or K+ currents but potently inhibition of high-voltage-activated (HVA) Ca2+ channels in DRG cells of rats. In order to study the structure-activity relationships of this toxin, the three-dimensional solution structure of HWTX-XVIIb1 was investigated by 2D 1H-NMR techniques. The complete sequence-specific assignments of proton resonance were obtained by analyzing a series of 2D spectra, including DQF-COSY, TOCSY and NOESY. All the backbone protons and more than 96% of the side-chain protons were identified by dαN, dβN, dNN and dαδconnectivities in NOESY spectrum. A series of constraint files were used for the final calculations of HWTX-VIIb1 by simulated annealing calculation using CNS program. A family of 20 accepted structures with lower energies and better Ramachandran plots were selected to represent the solution structure of HWTX-XVIIb1. The structures have no distance violations greater than 0.2? and no dihedral violations greater than 2.0°. The molecular structure of HWTX-XVIIb1 belongs to the inhibitor cystine knot (ICK) structural family with a short triple-stranded anti-parallelβ-sheet (Met8-Cys9, Cys19-Cys21 and Cys28-Cys30) and fourβ-turns (Lys4-Glu7, Cys9-Ile12, Cys14-Gly17 and Gly24-Arg27). These results provide a firm basis for the further study of the structure-activity relationships of HWTX-XVIIb1.