New Insectotoxin from Tibellus Oblongus Spider Venom Presents Novel Adaptation of ICK Fold.

The Tibellus oblongus spider is an active predator that does not spin webs and remains poorly investigated in terms of venom composition. Here, we present a new toxin, named Tbo-IT2, predicted by cDNA analysis of venom glands transcriptome. The presence of Tbo-IT2 in the venom was confirmed by proteomic analyses using the LC-MS and MS/MS techniques. The distinctive features of Tbo-IT2 are the low similarity of primary structure with known animal toxins and the unusual motif of 10 cysteine residues distribution. Recombinant Tbo-IT2 (rTbo-IT2), produced in E. coli using the thioredoxin fusion protein strategy, was structurally and functionally studied. rTbo-IT2 showed insecticidal activity on larvae of the housefly Musca domestica (LD100 200 µg/g) and no activity on the panel of expressed neuronal receptors and ion channels. The spatial structure of the peptide was determined in a water solution by NMR spectroscopy. The Tbo-IT2 structure is a new example of evolutionary adaptation of a well-known inhibitor cystine knot (ICK) fold to 5 disulfide bonds configuration, which determines additional conformational stability and gives opportunities for insectotoxicity and probably some other interesting features.

Effects of Selective Substitution of Cysteine Residues on the Conformational Properties of Chlorotoxin Explored by Molecular Dynamics Simulations.

Chlorotoxin (CTX) is a 36⁻amino acid peptide with eight Cys residues that forms four disulfide bonds. It has high affinity for the glioma-specific chloride channel and matrix metalloprotease-2. Structural and binding properties of CTX analogs with various Cys residue substitutions with l-α-aminobutyric acid (Abu) have been previously reported. Using 4.2 µs molecular dynamics, we compared the conformational and essential space sampling of CTX and analogs with selective substitution of the Cys residues and associated disulfide bonds with either Abu or Ser. The native and substituted peptides maintained a high degree of α-helix propensity from residues 8 through 21, with the exception of substitution of the Cys5⁻Cys28 residues with Ser and the Cys16⁻Cys33 residues with Abu. In agreement with previous circular dichroism spectropolarimetry results, the C-terminal ß-sheet content varied less from residues 25 through 29 and 32 through 36 and was well conserved in most analogs. The Cys16⁻Cys33 and Cys20⁻Cys35 disulfide-bonded residues appear to be required to maintain the αß motif of CTX. Selective substitution with the hydrophilic Ser, may mitigate the destabilizing effect of Cys16⁻Cys33 substitution through the formation of an inter residue H-bond from Ser16:OγH to Ser33:OγH bridged by a water molecule. All peptides shared considerable sampled conformational space, which explains the retained receptor binding of the non-native analogs.

Improved insecticidal activity of a recombinant baculovirus expressing spider venom cyto-insectotoxin.

Baculoviruses have a long history of safe use as specific, environmentally friendly insecticides that provide alternatives to chemical pesticides for controlling insect pests. However, their use has been limited by several factors, particularly their slow pathogenicity. In this study, we constructed a recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) and an Autographa californica multiple nucleopolyhedrovirus (AcMNPV) that expressed an insect-specific cyto-insectotoxin (Cit1a) from the venom of the central Asian spider Lachesana tarabaevi. Cit1a is a comparatively long linear cytolytic molecule that contains a predicted α-helix structure composed of two short membrane-acting antimicrobial peptides (MAMPs) that are joined together in a "head-to-tail" shape. Cit1a fused to polyhedrin gene (polh) (polh-cit1a) was expressed in the nuclei as polyhedra in silkworm larvae, Bm5 and Sf9 cells. An early death of Bm5 and Sf9 cells by recombinant BmNPV/Polh-Cit1a and AcMNPV/Polh-Cit1a was observed compared with control viruses that lacked the toxin gene. The infected cells showed a loss of cytoplasm, membrane integrity, and structural changes, suggesting that recombinant baculovirus-infected cells were killed by the necrosis caused by Cit1a. In addition, the BmNPV/Polh-Cit1a showed a significant reduction in the median lethal time (LT50) against silkworm larvae compared with those of control BmNPV that lacked the cit1a gene.