Anchoring of PI-PLC to DMPC Bilayers Involves Specific Cation-PI Interactions

Abstract

Phosphatidylinositol specific phospholipase Cs (PI-PLCs) from extra-cellular bacterial pathogens are associated with bacterial virulence. Membrane binding and enzymatic activity of many of these PI-PLCs is specifically enhanced by the presence of phosphatidylcholine (PC), an abundant phospholipid in the outer plasma membrane of eukaryotic cells targeted by these bacteria. In order to study the interactions of PI-PLC with the membrane, we first had to determine the orientation of the enzyme at the surface of a bilayer. This was achieved using an implicit membrane model (IMM1). To identify the molecular basis of membrane binding in these PI-PLCs, we then performed all-atom molecular dynamics (MD) simulations using the Bacillus PI-PLC structure and either a dimyristoylphosphatidylcholine (DMPC) or dimyristoylphosphatidylglycerol (DMPG) lipid bilayer. PI-PLC binding to DMPC bilayers was characterized by pi-cation interactions between the choline headgroups and a number of Trp and Tyr residues located in the B-helix, G-helix and active site rim loops, regions that have also been experimentally identified as important for PI-PLC membrane binding. Pi-cation occupancies for a single Trp or Tyr residue varied amongst the 6 PI-PLC copies in the MD simulations, consistent with experiments in which mutagenesis of a single aromatic residue in these regions had a limited impact on PI-PLC membrane binding and activity. A striking difference in the simulations using DMPG lipids is the absence of specific interactions mediated by the headgroups, which has an effect on the binding energy. Together, the simulations and experimental results suggest that PI-PLC binding to PC-rich membranes is mediated by aromatic-rich surface-accessible regions of the protein that engage in a myriad of transient pi-cation interactions with choline headgroups. This PC-binding mechanism may be utilized by other peripheral membrane proteins.