title: Controlling pore assembly of staphylococcal gamma-haemolysin by low temperature and by disulphide bond formation in double-cysteine LukF mutants.
abstract: Staphylococcal LukF and Hlg2 are water-soluble monomers of gamma-haemolysin that assemble into oligomeric pores on the erythrocyte membranes. Here, we have created double-cysteine LukF mutants, in which single disulphide bonds connect either the prestem domain and the cap domain (V12C-T136C, Cap-Stem), or two beta-strands within the prestem domain (T117C-T136C, Stem-Stem) to control pore assembly of gamma-haemolysin at intermediate stages. The disulphide-trapped mutants were inactive in erythrocyte lysis, but gained full haemolytic activity if the disulphide bonds were reduced. The disulphide bonds blocked neither the membrane binding ability nor the intermediate prepore oligomerization, but efficiently inhibited the transition from prepores to pores. The prepores of Cap-Stem were dissociated into monomers in 1% SDS. In contrast, the prepores of Stem-Stem were stable in SDS and had ring-shaped structures similar to those of wild-type LukF, as observed by transmission electron microscopy. The transition of both mutants from prepores to pores could even be achieved by reducing disulphide bonds at low temperature (2 degrees C), whereas prepore oligomerization was effectively inhibited by low temperature. Finally, real-time transition of Stem-Stem from prepores to pores on ghost cells, visualized using a Ca2+-sensitive fluorescent indicator (Rhod2), was shown by the sequential appearance of fluorescence spots, indicating pore-opening events. Taken together, these data indicate that the prepores are legitimate intermediates during gamma-haemolysin pore assembly, and that conformational changes around residues 117 and 136 of the prestem domain are essential for pore formation, but not for membrane binding or prepore oligomerization. We propose a mechanism for gamma-haemolysin pore assembly based on the demonstrated intermediates.
mesh_terms: D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D004910:Erythrocyte Membrane / Q000378:metabolism; D004912:Erythrocytes / Q000502:physiology; D015964:Gene Expression Regulation, Bacterial; D006460:Hemolysin Proteins; D006461:Hemolysis; D006801:Humans; D007956:Leukocidins / Q000737:chemistry / Q000235:genetics*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008854:Microscopy, Electron; D009154:Mutation; D021122:Protein Subunits; D013211:Staphylococcus aureus / Q000235:genetics / Q000378:metabolism; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004220:Disulfides; D006460:Hemolysin Proteins; D007956:Leukocidins; D008565:Membrane Proteins; D021122:Protein Subunits; C037956:gamma-hemolysin, Staphylococcus aureus; C074300:LukF protein, Staphylococcus aureus; D003545:Cysteine