Role of the Amino Latch of Staphylococcal α-Hemolysin in Pore Formation

Abstract

Staphylococcal ␣-hemolysin (␣HL) is a ␤ barrel pore-forming toxin that is secreted by the bacterium as a water-soluble monomeric protein. Upon binding to susceptible cells, ␣HL assembles via an inactive prepore to form a water-filled homoheptameric transmembrane pore. The N terminus of ␣HL, 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 ␣HL to form functional pores was diminished, but still substantial. We then discovered that the mutation Ser 217 3 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 ␣HL 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 ␣HL monomers in solution.

Assembly of the Bi-component Leukocidin Pore Examined by Truncation Mutagenesis

“…The N terminus of LukF is in a protease sensitive conformation, whereas the N terminus of LukS is protease-resistant. 2) In agreement with work on the ␣HL pore (see previous report (36)), the N termini of the LukF and LukS subunits are not required for pore formation. This was demonstrated in experiments where both LukF and LukS were truncated, as well as in experiments where a truncated subunit was assembled with its fulllength counterpart.…”

“…8A, stage 4). However, in the preceding report (36), we demonstrated that in various mutants of ␣HL the formation of a protease-resistant latch from Freshly translated LukF-S9C, LukS-D3C, and ␣HL-S3C labeled with 35 S[cysteine] were allowed to bind to rRBC membranes. In the cases of the leukocidins, the unlabeled WT counterpart was included to ensure pore formation.…”

“…The N termini are believed to reorganize during the insertion step to form a "latch" that reinforces the interactions between neighboring subunits (7,16). However, recent work from our laboratory suggests that, whereas this can occur, it is not required for pore formation in the case of ␣HL (Jayasinghe et al, preceding report (36)). Here, we use truncation mutagenesis to examine the contributions of the N and C termini of both LukF and LukS to the assembly of the leukocidin pore.…”

“…Additional evidence suggests that these proteins bind to membranes as monomers, associate on the membrane surface to form oligomeric prepores, and finally insert into the lipid bilayer (7,16,(21)(22)(23)) (see Fig. 1B in the preceding paper (36)). …”

Subunit Dimers of α-Hemolysin Expand the Engineering Toolbox for Protein Nanopores

“…This model for the linker assumes that the N terminus of one subunit latches tightly onto a second, as seen in the crystal structure of the WT heptamer. However, work in our laboratory has revealed that the N terminus of ␣HL is not critical for the formation of oligomers, and up to 17 residues can be deleted while retaining the ability to form pores (32). Furthermore, the related Vibrio cholerae cytolysin has no such N-terminal domain (33).…”