Homologous versus heterologous interactions in the bicomponent staphylococcal gamma-haemolysin pore.
Staphylococcal gamma-haemolysin HlgA-HlgB forms a beta-barrel transmembrane pore in cells and in model membranes. The pore is formed by the oligomerization of two different proteins and a still debated number of monomers. To clarify the topology of the pore, we have mutated single residues - placed near the right and left interfaces of each monomer into cysteine. The mutants were labelled with fluorescent probes, forming a donor-acceptor pair for FRET (fluorescence resonance energy transfer). Heterologous couples (labelled on complementary left and right interfaces) displayed a marked FRET, suggesting extensive HlgA-HlgB or HlgB-HlgA contacts. Heterologous control couples (with both components labelled on the same side) showed absent or low FRET. We found the same result for the homologous couple formed by HlgA [i.e. HlgA-HlgA in the presence of wt (wild-type) HlgB]. The homologous HlgB couple (HlgB-HlgB labelled on left and right interfaces and in the presence of wt HlgA) displayed a transient, declining FRET, which may indicate fast formation of an intermediate that is consumed during pore formation. We conclude that bicomponent pores are assembled by alternating heterologous monomers.
Viero
Gabriella
G
Istituto Trentino di Cultura (ITC) and Consiglio Nazionale delle Ricerche (CNR), Istituto di Biofisica, Sezione di Trento, Via Sommarive 18, I-38050 Povo (TN), Italy.
Cunaccia
Romina
R
Prévost
Gilles
G
Werner
Sandra
S
Monteil
Henri
H
Keller
Daniel
D
Joubert
Olivier
O
Menestrina
Gianfranco
G
Dalla Serra
Mauro
M
D019943:Amino Acid Substitution; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane; D002463:Cell Membrane Permeability; D003461:Crystallography; D004912:Erythrocytes / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006460:Hemolysin Proteins; D006461:Hemolysis; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D011817:Rabbits; D013211:Staphylococcus aureus / Q000378:metabolism*
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D006460:Hemolysin Proteins; C037956:gamma-hemolysin, Staphylococcus aureus