Paper Info
Title
Specificity of Loxosceles alpha clade phospholipase D enzymes for choline-containing lipids: Role of a conserved aromatic cage
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 alpha clade and the beta clade. Most alpha 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 structures of phospholipases D (PLDs) reveals a conserved aromatic cage in the alpha 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 alpha clade, Loxosceles intermedia alpha IA1 (Li_alpha IA) and Loxosceles laeta alpha III1 (Ll_alpha 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 alpha clade PLDs. Here, we confirmed that the i-face of alpha and beta 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 alpha clade PLDs. The MD simulation results are supported by in vitro liposome binding assay experiments. Author summaryEnvenomation following bites from recluse spiders (Loxosceles) causes loxoscelism, a necrotic tissue breakdown in mammals, and leads to skin degeneration and systemic reactions in the worst case. Recluse spiders belong to the Sicariidae family which also includes six-eyed sand spiders in the genera Sicarius and Hexopthalma. 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. Sphingomyelinase D/phospholipase D enzymes are one of the major toxins in venom of sicariid spiders, and have been divided in two clades called alpha and beta. The activity of alpha and beta clades toxins differs; most alpha clade toxins present high activity against lipids with choline headgroups (-N (CH3)(3)(+)) such as sphingomyelin, while activities in beta clade toxins vary and include preference for substrates containing ethanolamine headgroups (-NH3+). When comparing the structures of two alpha clade toxins and one beta clade toxin, we noticed the presence in the alpha clade toxins only of a cage consisting of three aromatic amino acids. In this work we used numerical molecular simulations to probe the role of this cage in the preference of alpha clade toxins for choline head groups over ethanolamine head groups.
Year
DOI
Venue
2022
10.1371/journal.pcbi.1009871
PLOS COMPUTATIONAL BIOLOGY
DocType
Volume
Issue
Journal
18
2
ISSN
Citations 
PageRank 
1553-734X
0
0.34
References 
Authors
0
8
Name
Order
Citations
PageRank
Emmanuel E Moutoussamy100.34
Qaiser Waheed200.34
Greta J Binford300.34
Hanif M Khan400.34
Shane M Moran500.34
Anna R Eitel600.34
Matthew H J Cordes700.34
Nathalie Reuter800.34