Interplay between Electrostatics and Cation-PI Interactions Governs the Specific Membrane Binding of Phosphatidylinositol-Specific Phospholipase-C

Abstract

Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (BtPI-PLC) is an amphitropic enzyme which cleaves GPI-anchored proteins off the outer surface of eukaryotic plasma membranes. Amphitropic proteins bind specifically and transiently to the surface of cell membranes, and their functions are regulated upon binding. It is commonly acknowledged that non-specific electrostatic forces are responsible for their long-range interactions with membranes. Using continuum electrostatics calculations we show how, despite having an overall negative charge (−7e), the charge distribution of BtPI-PLC leads to favorable electrostatic interactions with anionic membranes. However, the resulting electrostatic binding free energy, which is essential for membrane binding, is quite low. Mutation of a single, key basic residue to alanine diminishes this long range electrostatic contribution making it difficult for BtPI-PLC to associate with membranes. Once close to the membrane surface, short range non-specific hydrophobic interactions and specific cation-pi interactions with the N(Me)3 groups of phosphatidylcholine (PC) lipids of the membrane likely come into play for BtPI-PLC binding to the membrane surface. 500ns-long all-atom molecular dynamics simulations of BtPI-PLC docked to mixed bilayers with varying ratio of zwitterionic lipids indeed confirm this. Finally, we see that the interplay between long range electrostatics and short range, PC specific cation-pi interactions governs the specificity of BtPI-PLC for PC rich membrane. Moreover, our results show that BtPI-PLC can achieve favorable electrostatics interactions with lipid bilayers without having surface-exposed basic clusters suggesting that such clusters are not always necessary for the regulation of amphitropic enzyme binding.