Essential residues, W177 and R198, of LukF for phosphatidylcholine-binding and pore-formation by staphylococcal γ-hemolysin on human erythrocyte membranes

LukF and Hlg2 of staphylococcal gamma-hemolysin assemble into hetero-oligomeric pores on human red blood cells (HRBC). Here, we demonstrate, using a single-molecule imaging technique, that a W177T/R198T mutant of LukF, which exhibits no binding activity toward phosphatidylcholine, could form intermediate oligomers with Hlg2, including dimers, tetramers, and hexamer/heptamers, on HRBC. But, the mutant neither caused K+ efflux nor lysed HRBC, indicating that functional pores were not formed. Hence, we conclude that the W177 and R198 residues are essential for proper pore-formation by staphylococcal gamma-hemolysin. We also suggest that the interaction between the W177 and R198 residues, and phosphatidylcholine on membranes is the key to the formation of functional pores.