Specificity of Loxosceles α clade phospholipase D enzymes for choline-containing lipids: role of a conserved aromatic cage

Spider venom GDPD-like phospholipases D (SicTox) have been identified to be one of the major toxins in recluse spider venom. They are divided into two major clades: the clade and the {beta} clade. Most clade toxins present high activity against lipids with choline head groups such as sphingomyelin, while activities in {beta} clade toxins vary and include preference for substrates containing ethanolamine headgroups (Sicarius terrosus, St_{beta}IB1). A structural comparison of available PLDs structures reveals a conserved aromatic cage in the clade. To test the potential influence of the aromatic cage on membrane-lipid specificity we performed molecular-dynamics (MD) simulations of the binding of several PLDs onto lipid bilayers containing choline headgroups; two SicTox from the clade, Loxosceles intermedia IA1 (Li_IA) and Loxosceles laeta III1 (Ll_III1), and one from the {beta} clade, St_{beta}IB1. The simulation results reveal that the aromatic cage captures a choline-headgroup and suggest that the cage plays a major role in lipid specificity. We also simulated an engineered St_{beta}IB1, where we introduced the aromatic cage, and this led to binding with choline-containing lipids. Moreover, a multiple sequence alignment revealed the conservation of the aromatic cage among the clade PLDs. Here, we confirmed the membrane binding site of and {beta} clade PLDs on choline and ethanolamine-containing bilayers, respectively. Furthermore, our results suggest a major role in choline lipid recognition of the aromatic cage of the clade PLDs. The MD simulation results are supported by in vitro liposome binding assay experiments.