The leukocidin pore: evidence for an octamer with four LukF subunits and four LukS subunits alternating around a central axis

Abstract

The staphylococcal alpha-hemolysin (alphaHL) and leukocidin (Luk) polypeptides are members of a family of related beta-barrel pore-forming toxins. Upon binding to susceptible cells, alphaHL forms water-filled homoheptameric transmembrane pores. By contrast, Luk pores are formed by two classes of subunit, F and S, rendering a heptameric structure displeasing on symmetry grounds at least. Both the subunit stoichiometry and arrangement within the Luk pore have been contentious issues. Here we use chemical and genetic approaches to show that (1) the predominant, or perhaps the only, form of the Luk pore is an octamer; (2) the subunit stoichiometry is 1:1; and (3) the subunits are arranged in an alternating fashion about a central axis of symmetry, at least when a fused LukS-LukF construct is used. The experimental approaches we have used also open up new avenues for engineering the arrangement of the subunits of beta-barrel pore-forming toxins.

Figure 1.

Assembly of hetero-oligomeric pores. (A) Possible permutations of the subunits in Luk pores containing four LukF (yellow) and four LukS (red) subunits (see Electronic Supplemental Material; Table 1). (B) Interfaces between subunits in the Luk pore. An alternating arrangement of subunits (structure J in A) requires only two different interfaces between the LukF and LukS subunits (b-c and d-a, left). There are two interfaces, not one, because the subunits are asymmetric objects, not disks as shown. A random arrangement of subunits (structures A–J in A) requires four interfaces (b-c, d-a, b-a, d-c, right).

Figure 2.

Chemical cross-linking of adjacent subunits in the leukocidin octamer. (A) Cysteine mutagenesis for chemical cross-linking. Two adjacent subunits of the αHL pore are shown in ribbon form. The residues Arg-56 and Asp-2 were identified for cross-linking. The corresponding residues were identified as Lys-55 (Arg-56 in αHL; blue) in LukF (represented by yellow αHL subunit) and Asn-2 (Asp-2 in αHL; green) in LukS (represented by red αHL subunit). In αHL, the distance between the C_α atoms of Arg-56 and Asp-2 is ~11 Å. The model was generated with PyMOL version 0.97. (B) Sequence alignments of αHL, LukS, and LukF. (C) General scheme for the chemical cross-linking of Luk subunits. (A) LukF-K55C and LukS-N2C or LukS-N2C/Y113H are oligomerized on rabbit RBCMs (Fig. 3 ▶; Materials and Methods); the oligomer is purified by gel electrophoresis. (B) Cross-linking of adjacent subunits is carried out with Cu-OPA or SPAO; the arrow indicates a possible cross-link. (C) The noncovalent intersubunit interactions in the oligomer are disrupted by heating. (D) When desired, the cross-links in the resulting covalent dimers are broken with DTT to release the constituent monomers. (D) Mechanism of cross-linking of closely positioned cysteines with 4-sulfophenylarsine oxide (SPAO). (E) Possible cross-links in different arrangements of the Luk pores containing four LukF (yellow) and four LukS (red) subunits. A cross-link (arrow) involving the Cys residues in the mutants LukF-K55C and LukS-N2C can be formed only when LukS (red) precedes LukF (yellow) in a clockwise rotation, as viewed into the cap of the structure.

Figure 3.

Analysis of LukS-LukF dimers formed by cross-linking Luk oligomers. (A) Chemical cross-linking by Cu-OPA. LukF-K55C and LukS-N2C were oligomerized on rabbit RBCMs. Gel purified oligomers were treated with Cu-OPA (see Materials and Methods). The sample was divided into three equal portions, which were run on a 9% SDS–polyacrylamide gel. (Lane A) Unheated oligomer; (lane B) oligomer heated at 95°C for 5 min; (lane C) oligomer heated at 95°C for 5 min and then treated with DTT; (lane M) protein molecular weight markers (Amersham Biosciences). (B) Chemical cross-linking by SPAO. LukF-K55C and LukS-N2C/Y113H were oligomerized on rabbit RBCMs. Gel purified oligomers were treated with SPAO (see Materials and Methods). The sample was divided into three equal portions, which were run on a 10% SDS–polyacrylamide gel. (Lane A) unheated oligomer; (lane B) oligomer heated at 95°C for 5 min; (lane C) oligomer heated at 95°C for 5 min and then treated with DTT; (lane M) protein molecular weight markers. (C) IASD modification of residual uncross-linked monomers. LukF-K55C and LukS-N2C/Y113H were oligomerized on rabbit RBCMs. Gel-purified oligomers were treated with SPAO, heated at 95°C for 5 min and treated with IASD. (−) No IASD treatment; (+) treated with IASD.

Figure 4.

Formation of Luk oligomers beginning with different ratios of LukF and LukS subunits. (A) Five percent SDS–polyacrylamide gel. LukF and LukS oligomerized on RBCMs in different ratios: 15:1, 5:1, 1:1, 1:5, 1:15. (B) Analysis of the ratios of LukF to LukS in each band. Each band in A was gel-purified, heated and rerun on a 12.5% SDS–polyacrylamide gel. Phosphorimager quantification revealed the LukF: LukS ratio in each lane to be close to 1:1 as indicated.

Figure 5.

Experiments with a LukS-LukF fusion protein. (A) Gene construction. The 3′ end of the full-length gene of LukS (encoding residues 1–286; red) was linked to the 5′ end of the full-length gene of LukF (encoding residues 1–300; yellow) through a DNA sequence encoding a 15-residue serine/glycine linker (green). (B) Positions of the N and C termini of two adjacent subunits in the αHL heptamer. The N and C termini of LukF (yellow) and LukS (red) are presumed to assume similar positions as shown here. The model was generated by PyMOL version 0.97. (C) Hemolytic activity of the LukS-LukF fusion protein. (Row A) Wild type LukF with wild type LukS; (row B) LukS-LukF; (row C) LukS-LukF-BacTL; (row D) blank (no protein). Briefly, the proteins were serially two-fold diluted across the row. Pore formation was initiated by the addition of washed rabbit RBCMs and the decrease of light scattering was monitored (see Materials and Methods for details). (D) Oligomerization of LukS–LukF. Luk subunits were allowed to oligomerize on rRBCM. The products were run on a 10% SDS–polyacrylamide gel. (Lane A) Wild type LukS with wild type LukF; (lane B) LukS–LukF dimer; (lane C) LukS–LukF–BacTL; (lane M) protein molecular weight markers.

Figure 6.

Analysis of oligomers containing the LukS-LukF fusion protein. (A) Possible arrangements of the LukS-LukF dimer in the oligomer. (X) The two linked subunits are adjacent to each other (four dimers per oligomer). (Y) Only one subunit of each dimer participates in pore formation (eight dimers per oligomer). This is the extreme case of subunit exclusion from the central ring. (Z) The two linked subunits are in non-adjacent positions in the oligomer (four dimers per oligomer). (B) Possible permutations of subunits in pores formed from LukS-LukF and LukS-LukF-BacTL, assuming an alternating arrangement of F and S subunits. (C) Five percent SDS–polyacrylamide gel electrophoresis of oligomers formed on rRBCMs from various ratios of LukS-LukF and LukS-LukF-BacTL. (Lane A) 1:0 (lane B) 1:15; (lane C) 1:5; (lane D) 1:1; (lane E) 5:1; (lane F) 15:1; (lane G) 0:1. The five different bands are presumed to correspond to the five different permutations (P, Q, R, S, and T; Fig. 6B), as indicated. (D) Oligomer bands consist of LukS-LukF and LukS-LukF-BacTL. The oligomer bands in lanes A,F,D,B, and G of Figure 6C were heated to disrupt noncovalent intersubunit interactions and rerun on a 12.5% SDS–polyacrylamide gel. The two bands correspond to LukS-LukF and LukS-LukF-BacTL.

Figure 7.

Implications for engineering of βPFTs. (A) One or seven mutations can be introduced readily inside the αHL pore. (B) By mutating only one type of subunit, four mutations can be introduced in a symmetrical arrangement within the Luk pore. (C) By mutating both subunits, eight mutations (either the same or two different types) can be introduced inside the Luk pore. (D) With fused subunits (LukS-LukF), two different mutations can be introduced in adjacent subunits.