PT  - JOURNAL ARTICLE
AU  - Moutoussamy, Emmanuel E.
AU  - Waheed, Qaiser
AU  - Binford, Greta J.
AU  - Khan, Hanif M.
AU  - Moran, Shane M.
AU  - Eitel, Anna R.
AU  - Cordes, Matthew H.J.
AU  - Reuter, Nathalie
TI  - Specificity of <em>Loxosceles</em> α clade phospholipase D enzymes for choline-containing lipids: role of a conserved aromatic cage
AID  - 10.1101/2021.07.16.452673
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.07.16.452673
4099  - http://biorxiv.org/content/early/2021/07/18/2021.07.16.452673.short
4100  - http://biorxiv.org/content/early/2021/07/18/2021.07.16.452673.full
AB  - 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 PLDs structures 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 the membrane binding site of α and β 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 α clade PLDs. The MD simulation results are supported by in vitro liposome binding assay experiments.Competing Interest StatementThe authors have declared no competing interest.