I. Exploration of amphitropic protein interactions at the membrane interface; II. DNF2 - a plant protein with homology to bacterial PI-PLC enzymes

Amphitropic proteins, such as the virulence factor phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis, often depend on lipid-specific recognition of target membranes. However, the recognition mechanisms for zwitterionic lipids such as phosphatidylcholine (PC), which is enriched in the outer leaflet of eukaryotic cell membranes, are not well understood. Molecular dynamics (MD) simulation and mutagenesis results strongly indicate that PI-PLC interacts with PC head groups via cation-pi interactions with aromatic tyrosine residues, and suggest that cation-pi interactions at the interface may be a mechanism for specific lipid recognition by amphitropic and membrane proteins.;Aromatic amino acids can not only form cation-pi interactions at the interface but also insert into membranes and have hydrophobic interactions with lipid tails. Heretofore there has been no facile way to differentiate these two types of interactions. We show that specific incorporation of fluorinated amino acids into proteins can experimentally distinguish cation-pi interactions from membrane insertion of the aromatic side-chains. Fluorinated aromatic amino acids destabilize the cation-pi interactions by altering electrostatics of the aromatic ring while their enhanced hydrophobicity enhances membrane insertion. Incorporation of pentafluorophenylalanine or difluorotyrosine into a Staphylococcus aureus phosphatidylinositol-specific phospholipase C (PI-PLC) variant engineered to contain a specific PC-binding site demonstrates the effectiveness of this methodology. Applying this methodology to the plethora of tyrosine residues in Bacillus thuringiensis PI-PLC identifies those involved in cation-pi interactions with PC.;Cation-pi interactions provide a likely molecular mechanism for BtPI-PLC PC specificity but do not account for its preference for bilayers containing a small fraction of anionic lipids. MD simulations and fluorescence correlation spectroscopy (FCS) vesicle binding measurements of positively charged amino acids as well as surface tyrosine residues are used to formulate a complete model of BtPI-PLC specific binding to mixed anionic phospholipid/PC membrane.;DNF2, a new plant protein with homology to bacterial PI-PLC, is confirmed to be the first plant small PI-PLC enzyme that can cleave both PI and glycosylphosphatidylinositol (GPI) anchored proteins. GPI-anchored protein cleavage also confirms that DNF2 plays an important role in symbiosome, the intracellular compartment formed by the plant that contains nitrogen fixing bacteria.