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, which is enriched in the outer leaflet of eukaryotic cells, are not well understood. A 500 nanosecond long molecular dynamics simulation of PI-PLC at the surface of a lipid bilayer revealed a strikingly high number of interactions between tyrosines at the interfacial binding site and lipid choline groups with structures characteristic of cation-π interactions. Membrane affinities of PI-PLC tyrosine variants mostly tracked the simulation results, falling into two classes: (i) those with minor losses in affinity, Kd(mutant)/Kd(wildtype)≤5, and (ii) those where the apparent Kd was 50-200 times higher than wildtype. Estimating ΔΔG for these Tyr/PC interactions from the apparent Kd values reveals that the free energy associated with class I is ~1 kcal/mol, comparable to the value predicted by the Wimley-White hydrophobicity scale. In contrast, removal of class II tyrosines has a higher energy cost: ~2.5 kcal/mol towards pure PC vesicles. These higher energies correlate well with the occupancy of the cation-π adducts throughout the MD simulation. Together, these results strongly indicate that PI-PLC interacts with PC headgroups via cation-π interactions with tyrosine residues, and suggest that cation-π interactions at the interface may be a mechanism for specific lipid recognition by amphitropic and membrane proteins.

Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (BtPI-PLC) is a secreted virulence factor that binds specifically to phosphatidylcholine (PC) bilayers containing negatively charged phospholipids. BtPI-PLC carries a negative net charge and its interfacial binding site has no obvious cluster of basic residues. Continuum electrostatic calculations show that, as expected, nonspecific electrostatic interactions between BtPI-PLC and membranes vary as a function of the fraction of anionic lipids present in the bilayers. Yet they are strikingly weak, with a calculated ΔGel below 1 kcal/mol, largely due to a single lysine (K44). When K44 is mutated to alanine, the equilibrium dissociation constant for small unilamellar vesicles increases more than 50 times (∼2.4 kcal/mol), suggesting that interactions between K44 and lipids are not merely electrostatic. Comparisons of molecular-dynamics simulations performed using different lipid compositions reveal that the bilayer composition does not affect either hydrogen bonds or hydrophobic contacts between the protein interfacial binding site and bilayers. However, the occupancies of cation-π interactions between PC choline headgroups and protein tyrosines vary as a function of PC content. The overall contribution of basic residues to binding affinity is also context dependent and cannot be approximated by a rule-of-thumb value because these residues can contribute to both nonspecific electrostatic and short-range protein-lipid interactions. Additionally, statistics on the distribution of basic amino acids in a data set of membrane-binding domains reveal that weak electrostatics, as observed for BtPI-PLC, might be a less unusual mechanism for peripheral membrane binding than is generally thought.

Bacillus thuringiensis secretes the virulence factor phosphatidylinositol-specific phospholipase C (BtPI-PLC), which specifically binds to phosphatidylcholine (PC) and cleaves GPI-anchored proteins off eukaryotic plasma membranes. To elucidate how BtPI-PLC searches for GPI-anchored proteins on the membrane surface, we measured residence times of single fluorescently labeled proteins on PC-rich small unilamellar vesicles (SUVs). BtPI-PLC interactions with the SUV surface are transient with a lifetime of 379 ± 49 ms. These data also suggest that BtPI-PLC does not directly sense curvature, but rather prefers to bind to the numerous lipid packing defects in SUVs. Despite this preference for defects, all-atom molecular dynamics simulations of BtPI-PLC interacting with PC-rich bilayers show that the protein is shallowly anchored with the deepest insertions ∼18 Å above the bilayer center. Membrane partitioning is mediated, on average, by 41 hydrophobic, 8 hydrogen-bonding, and 2 cation−π (between PC choline headgroups and Tyr residues) transient interactions with phospholipids. These results lead to a quantitative model for BtPI-PLC interactions with cell membranes where protein binding is mediated by lipid packing defects, possibly near GPI-anchored proteins, and the protein diffuses on the membrane for ∼100–380 ms, during which time it may cleave ∼10 GPI-anchored proteins before dissociating. This combination of short two-dimensional scoots followed by three-dimensional hops may be an efficient search strategy on two-dimensional surfaces with obstacles.