Essential residues, W177 and R198, of LukF for phosphatidylcholine-binding and pore-formation by staphylococcal gamma-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.
Monma
Naota
N
Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan.
Nguyen
Vananh T
VT
Kaneko
Jun
J
Higuchi
Hideo
H
Kamio
Yoshiyuki
Y
D001120:Arginine / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry*; D002942:Circular Dichroism; D003603:Cytotoxins / Q000737:chemistry; D019281:Dimerization; D004910:Erythrocyte Membrane / Q000378:metabolism / Q000382:microbiology*; D004912:Erythrocytes / Q000378:metabolism / Q000382:microbiology*; D031541:Fluorescence Resonance Energy Transfer; D006460:Hemolysin Proteins / Q000737:chemistry*; D006461:Hemolysis; D006801:Humans; D007700:Kinetics; D007956:Leukocidins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000737:chemistry; D008958:Models, Molecular; D009154:Mutation; D054353:Perforin; D010713:Phosphatidylcholines / Q000737:chemistry*; D052899:Pore Forming Cytotoxic Proteins; D011188:Potassium / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D014364:Tryptophan / Q000737:chemistry
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D003603:Cytotoxins; D006460:Hemolysin Proteins; C503458:Hlg2 protein, Staphylococcus; D007956:Leukocidins; D008562:Membrane Glycoproteins; D010713:Phosphatidylcholines; D052899:Pore Forming Cytotoxic Proteins; C037956:gamma-hemolysin, Staphylococcus aureus; D054353:Perforin; C074300:LukF protein, Staphylococcus aureus; D014364:Tryptophan; D001120:Arginine; D011188:Potassium