Abstract
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 α clade and the β clade. Most α clade toxins present high activity against lipids with choline head groups such as sphingomyelin, while activities in β clade toxins vary and include preference for substrates containing ethanolamine headgroups (Sicarius terrosus, St_βIB1). A structural comparison of available structures of phospholipases D (PLDs) reveals a conserved aromatic cage in the α 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 α clade, Loxosceles intermedia αIA1 (Li_αIA) and Loxosceles laeta αIII1 (Ll_αIII1), and one from the β clade, St_βIB1. 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_βIB1, 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 α clade PLDs. Here, we confirmed that the i-face of α and β clade PLDs is involved in their binding to choline and ethanolamine-containing bilayers, respectively. Furthermore, our results suggest a major role in choline lipid recognition of the aromatic cage of the α clade PLDs. The MD simulation results are supported by in vitro liposome binding assay experiments.
Highlights
Sphingomyelinase D/phospholipase D (SMaseD/PLD) enzymes are one of the major toxins in venom of spiders in the family Sicariidae [1,2,3] including Loxosceles, the recluse spiders
Recluse spiders belong to the Sicariidae family which includes six-eyed sand spiders in the genera Sicarius and Hexopthalma
When comparing the structures of two α clade toxins and one β clade toxin, we noticed the presence in the α clade toxins only of a cage consisting of three aromatic amino acids
Summary
Sphingomyelinase D/phospholipase D (SMaseD/PLD) enzymes are one of the major toxins in venom of spiders in the family Sicariidae [1,2,3] including Loxosceles, the recluse spiders. The gene family comprising the sicariid SMaseD/PLD toxins is called SicTox reflecting evidence that this venom toxin is a synapomorphy for the family Sicariidae and is not a homolog of other PLDs [5]. Envenomation by recluse spiders causes necrotic tissue breakdown in mammals, a condition known as loxoscelism, and leads to skin degeneration and systemic reactions in the worst cases [2]. SicTox enzymes are sufficient causative agents for this syndrome in mammals [6] and are pharmaceutical targets [7,8]. While sicariid spiders are found natively on all continents except Australia, treatments of loxoscelism are typically antibody based and available in some regions of the Americas [9]
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