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Staphylococcal α-hemolysin (αHL) forms a heptameric pore that features a 14-stranded transmembrane β-barrel. We attempted to force the αHL pore to adopt novel stoichiometries by oligomerizing subunit dimers generated by in vitro transcription and translation of a tandem gene. However, in vitro transcription and translation also produced truncated proteins, monomers, that were preferentially incorporated into oligomers. These oligomers were shown to be functional heptamers by single-channel recording and had a similar mobility to wild-type heptamers in SDS-polyacrylamide gels. Purified full-length subunit dimers were then prepared by using His-tagged protein. Again, single-channel recording showed that oligomers made from these dimers are functional heptamers, implying that one or more subunits are excluded from the central pore. Therefore, the αHL pore resists all structures except those that possess seven subunits immediately surrounding the central axis. Although we were not able to change the stoichiometry of the central pore of αHL by the concatenation of subunits, we extended our findings to prepare pores containing one subunit dimer and five monomers and purified them by SDS-PAGE. Two half-chelating ligands were then installed at adjacent sites, one on each subunit of the dimer. Single-channel recording showed that pores formed from this construct formed complexes with divalent metal ions in a similar fashion to pores containing two half-chelating ligands on the same subunit, confirming that the oligomers had assembled with seven subunits around the central lumen. The ability to incorporate subunit dimers into αHL pores increases the range of structures that can be obtained from engineered protein nanopores.

Staphylococcal leukocidin (Luk) and alpha-hemolysin (alphaHL) are members of the same family of beta barrel pore-forming toxins (betaPFTs). Although the alphaHL pore is a homoheptamer, the Luk pore is formed by the co-assembly of four copies each of the two distantly related polypeptides, LukF and LukS, to form an octamer. Here, we examine N- and C-terminal truncation mutants of LukF and LukS. LukF subunits missing up to nineteen N-terminal amino acids are capable of producing stable, functional hetero-oligomers with WT LukS. LukS subunits missing up to fourteen N-terminal amino acids perform similarly in combination with WT LukF. Further, the simultaneous truncation of both LukF and LukS is tolerated. Both Luk subunits are vulnerable to short deletions at the C terminus. Interestingly, the N terminus of the LukS polypeptide becomes resistant to proteolytic digestion in the fully assembled Luk pore while the N terminus of LukF remains in an exposed conformation. The results from this work and related experiments on alphaHL suggest that, although the N termini of betaPFTs may undergo reorganization during assembly, they are dispensable for the formation of functional pores.

Staphylococcal alpha-hemolysin (alphaHL) is a beta barrel pore-forming toxin that is secreted by the bacterium as a water-soluble monomeric protein. Upon binding to susceptible cells, alphaHL assembles via an inactive prepore to form a water-filled homoheptameric transmembrane pore. The N terminus of alphaHL, which in the crystal structure of the fully assembled pore forms a latch between adjacent subunits, has been thought to play a vital role in the prepore to pore conversion. For example, the deletion of two N-terminal residues produced a completely inactive protein that was arrested in assembly at the prepore stage. In the present study, we have re-examined assembly with a comprehensive set of truncation mutants. Surprisingly, we found that after truncation of up to 17 amino acids, the ability of alphaHL to form functional pores was diminished, but still substantial. We then discovered that the mutation Ser(217) --Asn, which was present in our original set of truncations but not in the new ones, promotes complete inactivation upon truncation of the N terminus. Therefore, the N terminus of alphaHL cannot be critical for the prepore to pore transformation as previously thought. Residue 217 is involved in the assembly process and must interact indirectly with the distant N terminus during the last step in pore formation. In addition, we provide evidence that an intact N terminus prevents the premature oligomerization of alphaHL monomers in solution.