Structural characterisation of trimeric toxins from Australian snake venoms

JA Harrison1, C Kelso1, MP Padula2, GM Nicholson2 and JL Beck1

  1. School of Chemistry, University of Wollongong, Australia
  2. School of Life Sciences, University of Technology Sydney, Australia

Cytotoxic phospholipases A2 (PLA2) are a common feature of Australian snake venoms. Some of these proteins form heterogeneous trimers, which are composed of α (primary), β and γ (ancillary) subunits. The paucity of structural data for these toxins has limited investigations into the nature of structure-function relationships and cytotoxic activities. In the current work, nanoelectrospray ionisation mass spectrometry (nanoESI-MS) and ESI-ion mobility MS (IM-MS) were used to compare PLA2 from the venoms of Acanthophis antarcticus, Oxyuranus microlepidotus, Oxyuranus scutellatus and Tropidechis carinatus. This included confirmation of subunit composition, stoichiometries and cross-sectional areas. These results confirmed the presence of trimers (αβγ) with mass differences between isoforms arising from variations in the glycosylation of the γ subunit species. When collisional energies were applied the trimers dissociated into two species: β monomers and αγ assemblies. A comparison of these dissociation products to the subunits free in solution indicated that only the α and γ subunits lost charges (six positive) when assembled. This suggests that the β monomers are held in place predominantly by hydrophobic interactions, while the α and γ subunits are bound by electrostatic interactions. Finally, the data from IM-MS experiments suggest that these toxins have similar cross-sectional areas. This study points to structural similarities between trimeric PLA2 from Australian snakes and this information will be used as a basis to investigate the structure-function relationships of these toxins. Crystallography trials are in progress to confirm these observations.