Homologous versus heterologous interactions in the bicomponent staphylococcal γ-haemolysin pore¹

Staphylococcal γ-haemolysin HlgA–HlgB forms a β-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.