Cation−π Interactions As Lipid-Specific Anchors for Phosphatidylinositol-Specific Phospholipase C

Abstract

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.

Improving the Force Field Description of Tyrosine–Choline Cation−π Interactions: QM Investigation of Phenol–N(Me)4+ Interactions

“…10, 11 We had earlier reported cation-π interactions between phosphatidylcholine (PC) head groups of dimyristoylphosphatidylcholine lipids (DMPC) and tyrosine amino acids of a bacterial phospholipase; Bacillus thuringiensis phosphatidylinositol-specific phospholipase C ( Bt PI-PLC). 12 Cation-π adducts were also observed between tyrosine phenols and the DMPC choline groups in MD simulations with the additive CHARMM force field (CHARMM-ff). 13 Interestingly the occupancies of these interactions during the MD trajectory correlated qualitatively well with the effect the mutation of each of the tyrosines had on the experimentally measured affinity of the protein for DMPC vesicles.…”

Fluorinated Aromatic Amino Acids Distinguish Cation-π Interactions from Membrane Insertion

“…The targets of this enzyme are glycosylphosphatidylinositol-anchored proteins in the external leaflet of the plasma membrane, and affinity for PC would aid in directing the enzyme to its mammalian cell target. Mutagenesis (6), NMR (7), and molecular dynamics (MD) (6) simulations are consistent with choline cation-tyrosine complexes providing a significant amount of the binding energy for B. thuringiensis PI-PLC. However, none of these experiments absolutely distinguishes nonspecific side-chain insertion from very specific formation of a cation-complex with PC.…”

“…Details of the experimental set-up and data analysis have been previously described (6,17). Proteins were titrated with SUVs, and the fraction of protein bound to vesicles was determined from two species fits to the autocorrelations (obtained in cross-correlation mode),…”

“…6). Recent MD simulations have suggested transient cation-complexes exist when the protein is bound to a PC bilayer (6). Experimental tests to see which Tyr residues were critical for membrane binding substituted the Tyr with Ala.…”

“…However, the MD simulations strongly suggest this is not because of cation-interactions but rather arises from intramolecular interactions. Tyr-248 in the rim loop helps properly position Trp-242 (6), and this Trp is very important for membrane binding. All mutations to Tyr-248 appear to disrupt this interaction.…”