Specificity of Loxosceles α Clade Phospholipase D Enzymes for Choline-Containing Lipids: Role of a Conserved Aromatic Cage

Abstract

Phospholipase D (PLD) enzymes are one of the major toxins in the recluse spider (genus Loxosceles) venom. Loxosceles PLD enzymes are classified in either the α clade or the β clade, and some correlation exists between α or β clade membership and high or low catalytic activity against sphingomyelin (SM), respectively. Lajoie et al., recently showed that a β clade enzyme from Sicarius terrosus (St_βID1) had a strong preference for substrates containing ethanolamine headgroups, and suggested that this substrate specificity originated from the interactions between the PLD interfacial face and lipids. To further understand the substrate specificity of these enzymes, we performed simulations of two PLDs from the α clade, Loxosceles intermedia αIA1 (Li_αIA1) and Loxosceles laeta αIII1 (Ll_αIII1), and one from the β clade, St_βIB1i. Simulations were performed in the presence of two types of lipid bilayers, choline-containing and ethanolamine-containing bilayer. We observed that the two α clade PLDs bound to bilayers with choline-containing lipids (PC or SM) using the catalytic loop. By contrast, St_ βIB1i, did not bind to those bilayers. Analyses of the trajectories also reveal the importance, for the two α clade PLDs, of three aromatic residues located on the catalytic loop and forming a π-cage interacting with a choline group. A multiple sequence alignment of 18 PLDs of both clades reveal that the aromatic cage is conserved in α clade PLDs and absent from at least one major subgroup of the β clade enzymes. We suggest that this cage controls the affinity for choline headgroups, which is in agreement with earlier reports of PC lipid headgroups interacting with aromatic amino acids.