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. 2021 Jan 4;13(1):29.
doi: 10.3390/toxins13010029.

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

Yuliya Korolkova  1 , Ekaterina Maleeva  1 , Alexander Mikov  1   2 , Anna Lobas  3 , Elizaveta Solovyeva  3   4 , Mikhail Gorshkov  3 , Yaroslav Andreev  1   5 , Steve Peigneur  6 , Jan Tytgat  6 , Fedor Kornilov  7   8 , Vladislav Lushpa  7   8 , Konstantin Mineev  7   8 , Sergey Kozlov  1
Affiliations

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

Yuliya Korolkova et al. Toxins (Basel). .

Abstract

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.

Keywords: ICK fold; NMR structure; insectotoxin; proteome; spider venom; transcriptome.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Nucleotide sequence of one of the expressed sequence tags (EST) database clones, encoding the Tbo-IT2 peptide precursor. Navy blue highlights the signal peptide region, cyan—the propeptide fragment, green—the mature peptide, yellow—the post-translational modification.
Figure 2
Figure 2
The amino acid sequence of Tbo-IT2 toxin (shown in gray) and the corresponding proteolytic peptides (shown in black) detected in tryptic (top) and GluC (bottom) digests. The b- and y-fragments detected in the MS/MS spectra corresponding to the best PSMs for each peptide are shown in blue and red, respectively.
Figure 3
Figure 3
LC-MS/MS analysis of intact toxins from Tibellus oblongus spider venom. The intact Tbo-IT2 toxin was detected with 5 disulfide bonds and C-terminal amidation. (A) The extracted ion chromatogram of the most abundant isotope of Tbo-IT2 protein (841.55 m/z). (B) The mass-spectra of the intact Tbo-IT2 in four different charge states. The mass spectra of isotopic 13C cluster of intact Tbo-IT2 in (C) 5+ charge state and (D) 4+ charge state.
Figure 4
Figure 4
Dose-dependent insectotoxicity of rTbo-IT2 in comparison with ω-Tbo-IT1. The measurement was performed on a group of 12 house fly larvae for each dose (10, 25, 50, 100, 200 μg/g).
Figure 5
Figure 5
Overview of the NMR data that define the secondary structure of rTbo-IT2. Sequence, secondary structure (SS), NOE-connectivity, J-couplings, and hydrogen-deuterium exchange rate (H-D ex) are shown. Arrows indicate the β-strands of the β-sheet. Widths of the bars represent the relative intensity of cross-peaks in NOESY spectra. Colors of squares divide values of 3JHNHα into three groups: low, <6 Hz (white); medium, 6–8 Hz (grey); large, >8 Hz (black). Circles denote the HN protons, with the solvent exchange rates slower than 4 h−1.
Figure 6
Figure 6
Two-sided view on the rTbo-IT2. (A) The structure with the fewest restraint violation. Disulfide bonds are colored in orange, positively charged amino acid residues are in red, negatively charged residues are in cyan, and aromatic residues are in purple. (B) The best 10 structures out of the initial 100 are superimposed on the backbone of β-sheet residues. Disulfide bonds are colored in orange. (C) The contact surface of rTbo-IT2 is colored according to the hydrophobicity, from yellow (hydrophobic) to green (hydrophilic) using the White and Wimley scale [24].
Figure 7
Figure 7
Sequence alignment of Tbo-IT2 and other inhibitor cystine knot (ICK) peptides. Cysteine residues contributed to the ICK fold are highlighted in blue, additional cysteine residues are in red.
Figure 8
Figure 8
Comparison of Tbo-IT2 with other ICK peptides. (A) The primary structure alignment is based on spatial structure similarity with the SGTX1, robustoxin, purotoxin-1, and Magi3. The amino acid sequences are aligned according to the equivalent topological structure as determined by the PDBeFold algorithm. Residues that entered spatial alignment are shown in blue, whereas residues with a distance between Cα atoms more than 3 Å are shown in small letters. Similar disulfide bonds are shown in blue; additional disulfide bonds are shown in red (B) Spatial structure of Tbo-IT2, Cys17-Cys39, Cys22-Cys27 are shown in red; (C) Names and PDB codes of aligned peptides.
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