Structural venomics reveals evolution of a complex venom by duplication and diversification of an ancient peptide-encoding gene

Abstract

Spiders are one of the most successful venomous animals, with more than 48,000 described species. Most spider venoms are dominated by cysteine-rich peptides with a diverse range of pharmacological activities. Some spider venoms contain thousands of unique peptides, but little is known about the mechanisms used to generate such complex chemical arsenals. We used an integrated transcriptomic, proteomic, and structural biology approach to demonstrate that the lethal Australian funnel-web spider produces 33 superfamilies of venom peptides and proteins. Twenty-six of the 33 superfamilies are disulfide-rich peptides, and we show that 15 of these are knottins that contribute >90% of the venom proteome. NMR analyses revealed that most of these disulfide-rich peptides are structurally related and range in complexity from simple to highly elaborated knottin domains, as well as double-knot toxins, that likely evolved from a single ancestral toxin gene.

Keywords: proteomics; spider venom; structural venomics; transcriptomics; venom evolution.

Fig. 1.

Mass profile of H. infensa venom. (A) RP-HPLC chromatogram showing fractionation of crude H. infensa venom. Absorbance at 215 nm is shown in dark gray (left ordinate-axis) while mass count for each HPLC fraction is shown in light gray (right ordinate-axis). (B) Distribution of MALDI-TOF/TOF masses as a function of RP-HPLC retention time. (C and D) Histograms showing distribution and abundance of peptides in H. infensa venom as detected using (C) MALDI-TOF/TOF and (D) Orbitrap MS. Masses are grouped in 500-Da bins. Gray bars indicate cumulative total number of toxins (right ordinate-axis). (E) Euler plot showing overlap between mass counts generated via MALDI-TOF/TOF and Orbitrap MS. (F) A 3D landscape of H. infensa venom showing the correlation between RP-HPLC retention time and peptide mass and abundance generated by MALDI-TOF/TOF analysis. (Inset) A photo of a female H. infensa. Image credit: Bastian Rast (photographer).

Fig. 2.

Overview of the venom proteome of H. infensa. The venom proteome of H. infensa consists of 33 toxin superfamilies (SF1 to SF33). For each SF, the cysteine framework is shown in blue, and the 3D fold is classified as ICK, putative ICK, double ICK, non-ICK, or unknown. Light blue boxes enclose previously solved structures of superfamily members from H. infensa. Black boxes enclose structures of orthologous superfamily members from related mygalomorph spiders or, in the case of enzymes and CRiSP proteins, orthologs from venomous hymenopterans (bees and wasps). Red boxes enclose structures solved in the current study (SF6, SF22, SF23, and SF26). Stars enclosed by dashed black boxes signify toxin superfamilies for which no structural information is currently available. For each of the structures, β-strands are shown in blue, helices are green, and core disulfide bonds are shown as solid red tubes. For DRPs containing an ICK motif, additional disulfide bonds that do not form part of the core ICK motif are shown as orange tubes. Toxin superfamilies are named after gods or deities of death, destruction, and the underworld. For each structure shown, PDB accession numbers are given in the lower right corner.

Fig. 3.

Abundance of transcripts encoding DRPs and proteins obtained from sequencing of an H. infensa venom-gland transcriptome. Blue bars represent protein-encoding transcripts while red and gray bars denote transcripts encoding DRPs that do or don’t have an ICK scaffold, respectively. ICK transcripts dominate the venom-gland transcriptome. Superfamily numbers correspond to those shown in Fig. 2.

Fig. 4.

Structures of selected DRPs found in the venom of H. infensa, highlighting key structural innovations in “short” and “long” peptide toxins. (A) Schematic representation of the SF1 double-knot toxin, which is comprised of two independently folded ICK domains joined by an inflexible linker. (B) SF6 family peptides adopt a typical ICK fold with several unique elaborations, including an extended C-terminal tail that is stapled to the rest of the structure via an additional disulfide bond (“tail-lock”), an enlarged intercystine loop 4 that contains an α-helical insertion, and a highly extended N-terminal region that includes a six-residue α-helix and a short two-stranded β-sheet. (C) SF26 DRPs also adopt a highly elaborated knottin fold with a C-terminal tail tail-lock, a five-residue 3₁₀ helix in the core ICK region between Cys^II and Cys^III, and a long N-terminal extension that includes a short α-helix. (D) SF23 DRPs only differ from classical ICK toxins by having an extra disulfide bond (“β-sheet staple”) that stabilizes the β-hairpin loop. (E) Structural alignment of the ICK core regions of DRPs from SF1, SF6, SF17, SF22, and SF23, highlighting the strong conservation of the ICK motif (DDH in the case of SF22) regardless of the extent of structural elaborations outside this core region. (F) SF22 DRP represents a previously unidentified toxin fold comprised of two independent structural domains connected by a disulfide bond. The C-terminal domain forms a DDH core while the N-terminal domain adopts a DABS fold. In all panels, disulfide bonds comprising the ICK motif are shown as red tubes while additional noncore disulfide bonds are shown as orange tubes. β-sheets and α-helices are highlighted in blue and green, respectively.

Fig. 5.

Evolution of spider-venom ICK toxins. Maximum likelihood tree showing the phylogenetic relationship between H. infensa DRPs and DRPs isolated from the venom gland or other tissues of other spider species. The tree was rooted using the whip scorpion M. giganteus as the outgroup. Bootstrap values are shown at each node, except for nodes where support was 100. The tree shows that, although many of the phylogenetic relationships between superfamilies remain unresolved, all venom-derived DRPs form a well-supported monophyletic clade. Superfamily sequences belonging to H. infensa are highlighted in blue text, and representative structures for each superfamily are shown. DRP sequences from muscle and other tissues are highlighted in red; all other sequences (denoted by the light blue broken circle) represent venom DRPs. Venom peptides isolated from other species have their corresponding accession numbers/common toxin names listed in the labels, with the exception of the H. hainanum superfamily XVI clade. A summary of all accessions used can be found in SI Appendix.

Fig. 6.

Overview of structural innovations in spider-venom ICK peptides. Schematic overview of the mechanisms by which an ancestral ICK or DDH toxin was duplicated, conjugated, and elaborated upon to form the diversity of ICK scaffolds found in extant mygalomorph spider venoms. Gray numbered circles represent cysteine residues, with core disulfide bonds that form the cystine knot or DDH motif indicated by solid red lines. Additional noncore disulfides are highlighted in orange, with dashed lines indicating interdomain disulfide bonds. Blue arrows and green rectangles denote β-strands and α-helices, respectively. N and C termini are labeled, and PDB accession codes for representative structures are given below each schematic peptide fold.