Search for: All records

The glycerophosphodiester phosphodiesterase (GDPD)‐like SMaseD/PLD domain family, which includes phospholipase D (PLD) toxins in recluse spiders and actinobacteria, evolved anciently in bacteria from the GDPD. The PLD enzymes retained the core (β/α)₈barrel fold of GDPD, while gaining a signature C‐terminal expansion motif and losing a small insertion domain. Using sequence alignments and phylogenetic analysis, we infer that the C‐terminal motif derives from a segment of an ancient bacterial PLAT domain. Formally, part of a protein containing a PLAT domain repeat underwent fusion to the C terminus of a GDPD barrel, leading to attachment of a segment of a PLAT domain, followed by a second complete PLAT domain. The complete domain was retained only in some basal homologs, but the PLAT segment was conserved and repurposed as the expansion motif. The PLAT segment corresponds to strands β7–β8 of a β‐sandwich, while the expansion motif as represented in spider PLD toxins has been remodeled as an α‐helix, a β‐strand, and an ordered loop. The GDPD‐PLAT fusion led to two acquisitions in founding the GDPD‐like SMaseD/PLD family: (1) a PLAT domain that presumably supported early lipase activity by mediating membrane association, and (2) an expansion motif that putatively stabilized the catalytic domain, possibly compensating for, or permitting, loss of the insertion domain. Of wider significance, messy domain shuffling events can leave behind scraps of domains that can be salvaged, remodeled, and repurposed.

 

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.