Specific Transient Interactions Between a Bacillus Virulence Factor and Phosphatidylcholine in Membranes

Abstract

How do phospholipase Cs associated with bacterial virulence bind to membranes and search for substrates? The virulence factor Bacillus thuringiensis phosphatidylinositol‐specific phospholipase C (BtPI‐PLC) cleaves GPI‐anchored proteins off the outer leaflet of eukaryotic cells, helping the bacteria evade host defenses. Single molecule fluorescence microscopy studies of fluorescently labeled BtPI‐PLC interacting with model membranes reveal a koff of 2.64 ± 0.34 s‐1 at 22°C. This relatively fast dissociation is correlated with shallow anchorage of the protein in the membrane revealed by molecular dynamic simulations. Experimental and computational results suggest that cation‐π interactions between phosphatidylcholine (PC) headgroups and BtPI‐PLC Tyr residues play an important role in membrane binding. To further test this hypothesis, we substituted 3,5‐difluoroTyr for individual Tyr residues, thus perturbing cation‐π electrostatic interactions while increasing Tyr hydrophobicity. We also performed 31P NMR experiments with spin‐labeled BtPI‐PLC, to determine the location of PC binding sites on the protein surface. Both sets of experiments identify the same regions as important for PC binding. These biophysical and computational studies coupled with changes in enzyme activity provide a detailed, quantitative picture of how PC interactions with BtPI‐PLC influence its transient binding, PI cleavage and the search for substrates on the membrane.