Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (BtPI-PLC) is an amphitropic enzyme that 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 long-range interactions between proteins and membranes. Using continuum electrostatics calculations we show how, despite having an overall negative charge (-7e), the charge distribution of BtPI-PLC leads to a favorable although quite low electrostatic binding free energy with anionic membranes. The in silico mutation of a single, key basic residue to alanine diminishes this long-range electrostatic interaction explaining the significant decrease in the experimentally measured Kd. Multiple 500 ns-long all-atom molecular dynamics simulations of BtPI-PLC docked to mixed bilayers with varying ratios of zwitterionic lipids show that, 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 in the membrane come into play for BtPI-PLC binding to the membrane surface [1]. Comparing our simulation results with fluorescence correlation spectroscopy measurements of the membrane affinity of the wild-type enzymes and of various mutants, we conclude that the interplay between long-range electrostatics and short range, PC specific cation-pi interactions governs the specificity of BtPI-PLC for PC-rich membranes. Finally, we suggest that overlooked cation-pi interactions between membranes and aromatic amino acids of amphitropic proteins may play an important role not only in membrane binding but also in lipid specificity.

Investigating the forces governing the peripheral membrane association and dissociation of a bacterial phospholipase C (BtPI-PLC)

249th National Meeting (2015)

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