Sequence Determinants of Lipid Backbone Preference in Recluse Spider Toxins

Abstract

Envenomation by Sicariid spiders can lead to dermonecrosis, hemolysis, renal failure, and even death in humans. The toxin responsible for these symptoms is a sphingomyelinase D/phospholipase D (SMase D/PLD) that cyclizes two classes of lipids: sphingolipids and lysophospholipids. Known SMase D/PLD variants have activity against both lipid classes, making it difficult to elucidate the role of each activity in toxicity. Toxin variants with altered activity against only one of the two substrate types would help address this question, but the determinants of sphingolipid versus lysophospholipase preference are unknown. We used in silico docking to identify candidate sequence positions that may contribute to this preference, and then investigated the effects of single mutations at these positions using enzyme-coupled head-group release and 31P-NMR assays. We found that Ile198Thr or Thr198Ile mutations in a flexible loop adjacent to the active site have a ten-fold effect on sphingolipid activity but less than a two-fold effect on lysophospholipase activity. The mutations at position 198 should enable testing of whether it is the sphingolipase or lysophospholipase activity that is primarily responsible for the toxicity in vivo.