Structural venomics: evolution of a complex chemical arsenal by massive duplication and neofunctionalization of a single ancestral fold

Sandy Steffany Pineda, Yanni K-Y Chin, Eivind A.B. Undheim, Sebastian Senff, Mehdi Mobli, Claire Dauly, Pierre Escoubas, Graham M Nicholson, Quentin Kaas, John S Mattick, Glenn F King

Posted on: 14 December 2018 , updated on: 17 December 2018

Preprint posted on 3 December 2018

Spider venom disentangled: an interdisciplinary study reveals the complex composition of the Australian funnel-web spider venom and the large contribution of peptide families evolved to have a knotted structure

Selected by Tessa Sinnige

Categories: biochemistry


Spider venoms are known to contain a mixture of components including proteins and peptides, which have evolved for millions of years to target ion channels and receptors in order to immobilise prey. Many of the venom peptides are disulfide-rich peptides (DRP’s), and most DRP’s studied so far adopt an inhibitor cystine knot (ICK) fold in which several disulfide bonds create a knotted structure. These so-called ‘knottins’ have the advantage of being very stable and protease resistant. DRP’s can be highly variable in sequence outside of the cysteine residues required for the disulfide bonds, which has made it challenging to map the evolutionary process that gave rise to their striking diversity1.


Results of the preprint

In order to gain insights into the evolutionary history of DRP’s, the authors used a multi-omics and structural biology approach to study the venom from the Australian funnel-web spider Hadronyche infensa. Using MALDI and Orbitrap mass spectrometry on the crude venom they identified 3051 unique peptides, revealing a larger complexity than reported previously for different funnel-web spider species2,3. The authors note, however, that this could be due to improvements in sensitivity of the techniques. They then examined the venom composition in detail at the transcriptome level, and annotated and categorised the sequences to reveal 26 families of DRP’s, in addition to 7 protein families. Proteomics on trypsin-digested venom subsequently confirmed the presence of the majority of the predicted peptides and proteins.

The authors compared the features of the 26 identified DRP families to known structures and sequences, and could assign 12 of them to be knottins with a predicted ICK fold. Another 6 families were definitely not knottins based on homology to other known sequences, leaving 8 families of unknown structure and biological activity. The authors set out to solve the structures of representative DRP’s from these families by NMR spectroscopy, and were able to solve three structures that turned out to be knottins, albeit with different structural elaborations around the typical ICK fold. A fourth structure, on the other hand, represented a completely novel venom peptide fold. Figure 2 of the preprint shows all of the superfamilies with representative structures. The importance of the knottin DRP’s was illustrated by examining the relative expression levels of all the superfamilies now identified as such, adding up ~91% of the total venom peptidome.


Figure 2 from the preprint (reproduced under a CC-BY-NC-ND 4.0 International license) showing the peptide and protein superfamilies of the H. infensa venom, which have been given names of gods or deities of death, destruction and the underworld. Representative structures shown in red boxes were solved in the current study, the ones in cyan boxes were solved previously and the black boxes contain structures from orthologues.


Phylogenetic analysis suggested that the knottin superfamilies likely evolved from the same ancestral fold, although the precise phylogenetic relationship between the different families remained unclear. The phylogenetic tree indicated that either the fourth disulfide bond present in some families evolved independently on at least two occasions, or the ancestral fold contained four disulfide bonds, one of which was later on lost in a subset of families. The analysis showed conclusively that a gain of disulfide bonds occurred for some of the larger peptides, which acquired structural extensions around the knottin scaffold, ruling out that they resulted from gene duplication.

The preprint finally also provided some interesting insights into venom production. When the spider glands were depleted from venom by electrical stimulation, the gene expression pattern changed dramatically when examined three days later, now showing a large fraction of genes involved in cellular processes such as transcription and translation, as well as factors required for protein folding and particularly for the efficient formation of disulfide bonds.


What I like about this preprint

Biology never ceases to amaze me, and I was very excited to learn about the complexity of spider venom and the ways this might have evolved. Furthermore, it is great that the authors went all the way from proteomics and transcriptomics to detailed structure determination with really fascinating results – you don’t discover a bunch of new protein folds every day!



  1. First I have a small technical question: what could explain the limited overlap between the peptides identified with MALDI and Orbitrap, respectively?
  2. What would be required to map the phylogenetic relationships between the DRP superfamilies in more detail? Would it help to have more homologous sequences from other spider species?
  3. To what extent did the venom peptides co-evolve with their targets?
  4. With respect to therapeutic implications, could the knowledge about the composition and structural basis of spider venom be useful to improve antivenoms to treat bites? Or could spider venom peptides themselves have pharmaceutical applications?



(1) Rodríguez de la Vega, R. C. (2005) A note on the evolution of spider toxins containing the Ick-motif. Toxin Rev. 24, 383–395.

(2) Escoubas, P., Sollod, B., and King, G. F. (2006) Venom landscapes: Mining the complexity of spider venoms via a combined cDNA and mass spectrometric approach. Toxicon 47, 650–663.

(3) Palagi, A., Koh, J. M. S., Leblanc, M., Wilson, D., Dutertre, S., King, G. F., Nicholson, G. M., and Escoubas, P. (2013) Unravelling the complex venom landscapes of lethal Australian funnel-web spiders (Hexathelidae: Atracinae) using LC-MALDI-TOF mass spectrometry. J. Proteomics 80, 292–310.

Tags: inhibitor cystine knot, nmr spectroscopy, phylogenetics, proteomics, toxins, transcriptomics


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