orals 5th Asia-Pacific NMR Symposium 2013

Tracking the evolutionary journey of spider toxins using high-throughput NMR methods (#89)

Yanni KY Chin 1 , Sandy S Pineda 1 , Sebastian Senff 1 , Mehdi Mobli 1 2 , Glenn F King 1
  1. Institute for Molecular Bioscience, University of Queensland, St Lucia, QLD, Australia
  2. Centre of Advanced Imaging, University of Queensland, St Lucia, QLD, Australia

Spiders are among the most successful predators in the animal kingdom.  Their success is due in large part to the evolution of pharmacologically complex venoms that play important roles in defense, predation, and competitor deterrence. The major components of most spider venoms are disulfide-rich peptides of mass 3–9 kDa. Almost all spider-toxin structures reported to date are variants of a three-disulfide scaffold known as the inhibitor cystine knot (ICK) motif. It has been proposed that the ICK motif is an elaboration of a simpler two-disulfide ancestral fold named the disulfide-directed β-hairpin (DDH). However, given the small number of spider-toxin structures available, it is difficult to draw definitive conclusions about the range of 3D folds present in spider venoms and their evolutionary history. Thus, we decided to survey the complete structural landscape of a single spider venom.

Considering the number of toxin structures required to adequately describe a venom landscape, rapid structure determination techniques were essential for this study. Thus, we developed an efficient bacterial expression system for production of 13C/15N-labelled toxins and acquired 3D/4D NMR experiments using non-uniform sampling. This greatly reduced acquisition times and improved resolution in the indirect dimensions, thus facilitating automated spectral assignment and structure calculation. High-quality toxin structures can now be determined in less than one week.

A comprehensive structural analysis of toxins found in the venom of the lethal Australian funnel-web spider Hadronyche infensa revealed several novel structural scaffolds, including the first example in spider venoms of the proposed ancestral DDH motif. This study suggests that the remarkable pharmacological diversity of spider venoms has evolved by massive duplication and subsequent neofunctionalisation of a single ancestral DDH gene.