Homologous versus heterologous interactions in the bicomponent staphylococcal γ-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 oil 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 oil 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.