Stochastic assembly of two-component staphylococcal gamma-hemolysin into heteroheptameric transmembrane pores with alternate subunit arrangements in ratios of 3:4 and 4:3.
Self-assembling, pore-forming toxins from Staphylococcus aureus are illustrative molecules for the study of the assembly and membrane insertion of oligomeric transmembrane proteins. On the basis of previous studies, we have shown that the two-component gamma-hemolysin assembles from LukF (or Hlg1, 34 kDa) and Hlg2 (32 kDa) to form ring-shaped transmembrane pores of ca. 200 kDa. Here we show that LukF and Hlg2 assemble in a stochastic manner to form alternate complexes with subunit stoichiometries of 3:4 and 4:3. High-resolution electron microscopic images of negatively stained pore complexes clearly revealed a heptameric structure. When adjacent monomers in the pore complexes were randomly cross-linked by using glutaraldehyde, LukF-LukF, LukF-Hlg2, and Hlg2-Hlg2 dimers were detected in an approximate ratio of 1:12:1, suggesting that LukF and Hlg2 were alternately arranged in the pore complex in molar ratios of 3:4 and 4:3. The alternate arrangements of LukF and Hlg2 in molar ratios of 3:4 and 4:3 were also visualized under electron microscope with the pore complexes consisting of glutathione S-transferase fusion protein of LukF or Hlg2 and wild-type protein of Hlg2 or LukF, respectively.
Sugawara-Tomita
Noriko
N
Department of Molecular and Cell Biology, Graduate School of Agricultural Science, Tohoku University, Aoba-ku, Sendai 981-8555, Japan.
Tomita
Toshio
T
Kamio
Yoshiyuki
Y
D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000737:chemistry*; D006460:Hemolysin Proteins; D007956:Leukocidins / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry; D008854:Microscopy, Electron; D008970:Molecular Weight; D021122:Protein Subunits
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D007956:Leukocidins; D008565:Membrane Proteins; D021122:Protein Subunits; C037956:gamma-hemolysin, Staphylococcus aureus; C074300:LukF protein, Staphylococcus aureus