Assembly mechanism of the α-pore-forming toxin cytolysin A from Escherichia coli

Abstract

The cytolytic toxin cytolysin A (ClyA) from Escherichia coli is probably one of the best-characterized examples of bacterial, α-pore-forming toxins (α-PFTs). Like other PFTs, ClyA exists in a soluble, monomeric form that assembles to an annular, homo-oligomeric pore complex upon contact with detergent or target membranes. Comparison of the three-dimensional structures of the 34 kDa monomer and the protomer in the context of the dodecameric pore complex revealed that ClyA undergoes one of the largest conformational transitions described for proteins so far, in which 55% of the residues change their position and 16% of the residues adopt a different secondary structure in the protomer. Studies on the assembly of ClyA revealed a unique mechanism that differs from the assembly mechanism of other PFTs. The rate-liming step of pore formation proved to be the unimolecular conversion of the monomer to an assembly-competent protomer, during which a molten globule-like off-pathway intermediate accumulates. The oligomerization of protomers to pore complexes is fast and follows a kinetic scheme in which mixtures of linear oligomers of different size are formed first, followed by very rapid and specific association of pairs of oligomers that can directly perform ring closure to the dodecameric pore complex.This article is part of the themed issue 'Membrane pores: from structure and assembly, to medicine and technology'.

Keywords: assembly kinetics; assembly of membrane complexes; cytolysin A; α-pore-forming toxins.

Figure 1.

Three-dimensional structures of the soluble ClyA monomer and the ClyA protomer (a) and the ClyA pore complex (b). (a) Comparison of the soluble monomer (left, pdb ID 1QOY) and the protomer (right, pdb ID 2WCD). In the monomer, the N-terminal helix (residues 1–47) is coloured light blue, the short helices in the head domain (residues 163–180 and 196–200) are coloured dark blue and the β-hairpin (residues 185–195) is coloured yellow. The four remaining helices of the tail domain are depicted in red, loops are shown in green. The colour code for residues in the protomer is the same as that in the monomer. Note the β-to-α and loop-to-α transitions upon protomer formation. (b) The dodecameric ClyA pore (pdb ID 2WCD) in side view (left), top view (upper right) and bottom view (lower right). One protomer is coloured according to (a). The membrane-spanning region of ClyA is indicated in the side view (grey band). The dimensions of the pore complex are indicated, as well as the inner diameter of the pore at its narrowest opening in the membrane-spanning region. Note that the largest part of the pore complex is extracellular.

Figure 2.

Positions of the FRET pair AlexaFluor 488 (donor, green) and AlexaFluor 594 (acceptor, red) with which ClyA was labelled at positions 56 and 252, respectively, for single-molecule FRET experiments. The C_β-C_β distances (d) of the cysteines introduced at positions 56 and 252 for site-specific labelling are indicated for the monomer (top left), protomer (bottom left) and the pore complex (right). The pore is tilted by 10° relative to the straight bottom view orientation (figure 1b) for best visualization of the positions of Cys56 inside the pore and Cys252 on the outer surface of the pore complex. Note that donor/acceptor–labelled ClyA was mixed with excess wild-type ClyA, so that pore complexes statistically only contained a single, labelled protomer. The colour code of secondary structures is the same as that in figure 1.

Scheme 1.

DDM-induced protomer formation from ClyA monomers is slowed by formation of the off-pathway intermediate I.

Scheme 2.

Only two rate constants are required to describe the kinetics of pore (P₁₂) assembly from protomers in the concentration range of 5 nM–5 µM.

Figure 3.

Mechanism of pore complex formation from assembly-competent protomers (filled circles). The entire pore assembly process could be approximated and globally fitted over a wide range of concentrations with only two rate constants: a single rate contant (k_elong.) for dimerization and any reaction that leads to larger oligomers (a), and a single rate constant (k_pore) for the association of any pair of oligomers that directly yields the dodecameric pore complex (b). The thick arrows in the lower panel indicate that pore formation by association of oligomers of similar size occurs more frequently than pore formation from oligomers of different size. Note that the indicated, pore-like curvature of linear oligomers is only tentative, but supported by the 300-fold higher value of k_pore compared with k_elong. (scheme 2). (Online version in colour.)

Figure 4.

Positions of the engineered cysteine pairs in the redox switch variants ClyA CC6/264 (a) and CC50/190 (b). Formation of an intramolecular disulfide bond between each cysteine pair in the respective, soluble monomer (top panels) prevents the conformational transition to assembly-competent protomers (bottom panels) in detergent or target membranes. The engineered disulfide bond in the monomer is highlighted by a black dashed box, and depicted in detail (top right panels). The positions of the introduced cysteines in the modelled structures of the reduced protomers are indicated by yellow spheres. The crystal structure of the disulfide-bonded monomer of ClyA CC6/264 was solved (pdb ID 4PHO), while the structure of the oxidized monomer of ClyA CC50/190 was modelled based on the structure of the soluble ClyA wild-type monomer (pdb ID 1QOY).

Scheme 3.

Kinetic mechanism of DDM-induced protomer formation in ClyA_ox with a disulfide bond between the natural cysteine pair Cys87/Cys285 (top), and free energy differences (in kJ mol⁻¹) between the monomer, intermediate and protomer for both ClyA redox states.