Pore Forming Protein Toxins

Many organisms produce polypeptide chains that can exist either as a water-soluble state or as a membrane-embedded species, usually an oligomeric integral membrane protein (Gouaux 1997; Heuck et al. 2001). The conversion between both protein forms is spontaneous, and it is generally accepted that it proceeds through discrete steps: water-soluble state, membrane-receptor binding, oli-gomerization, membrane insertion and final formation of the functional pore. The current structural information on these pore-forming proteins (PFPs) demonstrates that the above conversion entails concerted large-scale conformational changes which raises numerous questions, nowadays only partially understood, which connect not only with protein structure but also with protein folding and dynamics.

Despite their structural diversity (Parker and Feil, 2005), PFPs are usually classified into two types on the basis of the putative structure of the transmembrane (TM) domain: a-PFP, which are predicted to insert amphipathic a-helices, and P-PFP, which utilize amphipathic P-hairpins to form a membrane-spanning P-barrel. Regarding PFPs with known three-dimensional structure, the group of a-PFPs includes colicins (Wiener et al. 1997; Stroud et al. 1998), exotoxin A from Pseudomonas aeruginosa (Allured et al. 1986), insecticidal crystal proteins Cry3A (Li et al. 1991) and CrylAa (Grochulski et al. 1995) from Bacillus thuringiensis, and diphteria toxin from Corynebacterium diphteriae (Choe et al. 1992) (Fig. 3.9).

On the other hand, the group of P-PFP, includes aerolysin from Aeromonas hy-drophyla (Parker et al. 1994), e-toxin from Clostridium septicum (Cole et al. 2004), hemolytic lectin LSL from the mushroom Laetiporus sulphureus (Mancheno et al. 2005), crystal protein Cyt2A from B. thuringiensis (Li et al. 1996), a-hemolysin from Staphylococcus aureus (Song et al. 1996), perfringolysin O from C. perfrin-gens (Rossjohn et al. 1997), and the anthrax toxin produced by Bacillus anthracis which is composed of anthrax protective antigen (PA) (Petosa et al. 1997), directly involved in the formation of heptameric pores in the target membrane, and the enzymatic domain lethal factor (LF) and edema factor (EF). Remarkably, in this last case, the crystal structure of the complex between PA and its host cell receptor has been recently solved for the first time (Santelli et al. 2004) (Fig. 3.10).

In addition to the above-mentioned a-PFPs and P-PFPs, all coming from prokaryotic sources, the crystal structures of two eukaryotic toxins from sea anemones have recently been solved, those of equinatoxin II from Actinia equina (Athanasiadis et al. 2001) and sticholysin II from Stichodactyla helianthus (Man-cheno et al. 2003). In this last case, the determination of the low-resolution structure of a tetrameric assembly of sticholysin II by means of electron microscopy analyses of 2D crystals of the toxin formed on lipid monolayers suggested a model for the functional pore, which is consistent with the formation of toroidal membrane pores (Fig. 3.11).

In Figs. 3.9 and 3.10, a gallery of the crystal structures of the above mentioned PFPs can be observed; these reveal the broad diversity of structural protein architectures present in the hitherto poorly understood PFPs realm. In the following, we present a brief overview of the structural basis of the mechanism of the biological activity of a deeply analysed member of each group of PFPs: colicin la from the group of a-PFPs, a-hemolysin from the P-PFPs, and also recent results from the eukaryotic actinoporins.

Pore Protein

Colicin la

Exotoxin A

Diphteria toxin

Fig.3.9. Ribbon representations of a-PFPs. Regions directly involved in pore-formation are indicated in green. For clarity, the scales of the different proteins are not comparable. PDB codes are: (a) 1CII for colicin la from E. coli, (b) 1IKQ for exotoxin A from Pseudomonasaeruginosa, (c) 1DLC for Cry3A and (d) 1CIY for CrylAa, both from B. thuringiensis, and (e) 1XDT for diphteria toxin from Corynebacterium diphteriae. Figures were prepared with the program PyMOL

Anthrax Pore Formation

Aerolysin e-toxin ot-hemolysin

Perfringolysin 0

Anthrax protective antigen

Fig. 3.10. Ribbon representations of (3-PFPs. Domains responsible for pore-formation are highlighted in green. PDB codes are: 1PRE for aerolysin from Aeromonas hydrophyla, 1UYJ for e-toxin from Clostridiumsepticum, 1W3Afor the hemolytic lectin LSL from the mushroom Laet-iporussulphureus, ICBYfor Cytbfrom B. thuringiensis, 7AHLforthe oligomeric pore of a-hemo-lysin from Staphylococcus aureus, 1PFO for perfringoiysin 0 from C. perfringens, and 1ACC for anthrax protective antigen from Bacillus anthracis. Figures were prepared with the program PyMOL

Colicins are a-PFPs produced by several strains of E. coli which act on closely related bacteria (Stroud et al. 1998). The plasmid responsible for the production of colicins also provides the colicin-producing cells with an immunity system which protects them against their own toxins. Colicins present distinct structural domains arranged along the primary structure, corresponding to specific functional domains (Fig. 3.12): receptor binding (R-domain), translocation across the outer membrane (T-domain), and cytotoxic (membrane pore-formation, DNAse, RNAse, etc.; C-domain). The first step in their biological activity is binding to an outer membrane receptor through the R-domain, which is a porin involved in active transport, usually composed of a 12-14 0-stranded TM barrel. Then, to gain access to the plasma membrane, colicins parasite either the Tol or the Ton pathways of transport through the periplasm. After spanning the periplasm, the C-terminal domain is translocated across the outer membrane and finally reaches the plasma membrane.

Colicin la is an extremely elongated molecule (-210 A), in which two long a-helices (T3 and CI) segregate the R-domain from the T- and C-domains (Fig. 3.12) (Wiener et al. 1997). The R-domain binds to the porin Cir which is involved in the transport of iron-chelated siderophores (Stroud et al. 1998). Once the receptor has been recognized, colicin la uses the integral plasma membrane TonB protein to gain access to the plasma membrane. In this sense, colicin la has a

Porin And Tonb Proteins

POC binding site Prepore Functional pore

Fig. 3.11. Ribbon model of the colicin la molecule (PDB code: 1CII) indicating the different functional domains: R-domain (violet), which is Involved In receptor binding;T-domain (violet), which directly participates In protein translocation across the outer membrane, and C-domaln or channel forming domain (green). Hydrophobic helices a8 and a9, which are sandwiched by eight amphipathlc helices, are Indicated. Figures were prepared with the program PyMOL

POC binding site Prepore Functional pore

Fig. 3.11. Ribbon model of the colicin la molecule (PDB code: 1CII) indicating the different functional domains: R-domain (violet), which is Involved In receptor binding;T-domain (violet), which directly participates In protein translocation across the outer membrane, and C-domaln or channel forming domain (green). Hydrophobic helices a8 and a9, which are sandwiched by eight amphipathlc helices, are Indicated. Figures were prepared with the program PyMOL

Fig. 3.12. Three-dimensional structures of the Staphylococcus aureus a-hemolysln (a) hep-tamerlc pore, and that of the (b) water-soluble homologue LukF (PDB code: 2LKF), as ribbon models. Comparison of both structures reveals a mechanism of pore-formation involving large-scale conformational changes essentially In two regions: the N-termlnal segment latch (violet) and the stem {green). Figures were prepared with the program PyMOL

Fig. 3.12. Three-dimensional structures of the Staphylococcus aureus a-hemolysln (a) hep-tamerlc pore, and that of the (b) water-soluble homologue LukF (PDB code: 2LKF), as ribbon models. Comparison of both structures reveals a mechanism of pore-formation involving large-scale conformational changes essentially In two regions: the N-termlnal segment latch (violet) and the stem {green). Figures were prepared with the program PyMOL

hydrophobic stretch near the N-terminal end called the TonB box, which is conserved in TonB-dependent colicins and receptors participating in the TonB pathway, and which is required for the interaction with TonB. The length of a-helices T3 and Cl (-160 Â) permits colicin la to span the periplasm (average width 150 Â), while interacting both with the receptor and the inner membrane. Final channel formation in the plasma membrane of the target cell requires the translocation of the C-domain, which is formed by two hydrophobic helices surrounded by eight additional amphipathic a-helices (Fig. 3.12). The C-domain of colicin la interacts best with acidic rather than neutral phospholipids, indicating that electrostatic interactions are important in the initial steps of membrane association (Elkins et al. 1997). This electrostatically driven association is followed by insertion into the lipid bilayer which is driven by hydrophobic interactions (Za-kharovet al. 1996; Heymann et al. 1996). It must be remarked that despite the very low sequence identity between the C-domains from colicins la, A and El, they share the same C-domain scaffold (Parker et al. 1996; Elkins et al. 1997; Wiener et al. 1997), which suggests this helical domain is an efficient membrane-inserting motif. The current model, explaining membrane penetration by pore-forming colicins, is the umbrella model initially proposed for colicin A (Parker et al. 1990), with minor modifications (Parker and Feil, 2005). This mechanism involves the penetration of the hydrophobic a-helices, after previous opening of the domain. The conformational transition between the different states may be facilitated by the existence of a molten globule intermediate induced at low pH conditions (van der Goot et al. 1991; Muga et al. 1993).

Without doubt, a-hemolysin from S. aureus predominates the scenario of the P-PFPs as it represents the first and so far unique P-PFP oligomeric state whose high-resolution three-dimensional structure has been solved (Song et al. 1996). Comparison of this structure with those of the water-soluble states of various a-hemolysin homologues, LukF (Olson et al. 1999), LukF-PV (Pèdelacq et al.

1999), is of special interest for understanding not only the structural basis of the pore-formation mechanism by P-PFPs, but also the folding and structure of integral membrane proteins containing TM barrels (Montoya and Gouaux 2003). This structural information, together with other experimental evidence, points to a complex mechanism of a-hemolysin pore-formation, which may proceed through several discrete conformational states: water-soluble, membrane-bound monomer, heptameric prepore, and final functional inserted heptameric pore. Although the crystal structure of the water-soluble states of the a-hemolysin homologues LukF (Olson et al. 1999) (Fig. 3.13) and LukF-PV (Pedelacq et al. 1999) exhibit close resemblances with the overall structure of the a-hemolysin protomer, there are two regions that adopt dramatically different conformations when these states are compared; the so-called amino latch and the pre-stem regions suffer large-scale conformational changes upon pore formation (see below) (Fig. 3.13b).

Fig. 3.13. (a) Crystal structure of the pore-forming toxin Stnll from the anemone Stichodactyla helianthus (PDB code: 1GWY), and a (d) snapshot of the phosphocholine-binding site (PDB code: 1072), indicating residues involved in lipid-headgroup. (b) and (e) The electron microscopy envelope clearly revealed the existence of tetrameric species, and also conformational changes in the N-terminal a-helix of Stnll. (c) and (f) A putative model for the functional pore in which the N-terminal region adopts an extended helical conformation is also shown. Figures were prepared with the programs MOLSCRIPT and PyMOL

Fig. 3.13. (a) Crystal structure of the pore-forming toxin Stnll from the anemone Stichodactyla helianthus (PDB code: 1GWY), and a (d) snapshot of the phosphocholine-binding site (PDB code: 1072), indicating residues involved in lipid-headgroup. (b) and (e) The electron microscopy envelope clearly revealed the existence of tetrameric species, and also conformational changes in the N-terminal a-helix of Stnll. (c) and (f) A putative model for the functional pore in which the N-terminal region adopts an extended helical conformation is also shown. Figures were prepared with the programs MOLSCRIPT and PyMOL

The crystal structure of the heptameric a-hemolysin pore (Song et al. 1996), reveals a mushroom-shaped assembly (Fig. 3.13), with three distinct regions: stem, rim and cap. The oligomeric complex measures approximately 100 A in height and up to 100 A in diameter, with an inner solvent-filled tunnel parallel to the seven-fold axis, which ranges from -15 to -46 A in diameter. The stem or transmembrane region is composed of a 14-stranded P-barrel which results from the association of seven P-hairpins, each one contributed from a single monomer. This folding may indicate that the P-hairpin constitutes the basic structural unit of TM P-barrels which in fact agrees with current studies on constitutive TM P-barrels (Wimley 2003). Another evident structural feature of the stem region, also typically found in constitutive TM P-barrels (Schulz 2000; Wimley 2003), is that it is formed by an even number of strands with an alternating pattern of hydrophobic and small and polar residues. Whereas hydrophobic residues are oriented toward the hydrophobic core of the membrane, polar residues constitute the walls of the barrel lumen. On the other hand, the rims are the protein regions located in the close vicinity of the membrane outer interface. A characteristic feature of these regions, also in common with porins (Wimley 2003), is the presence of a high concentration of aromatic residues, which are known to exhibit a high affinity for membrane interfaces (Killian and von Heijne 2000), and also the presence of basic residues which would interact with the phospholipid headgroups. Finally, the cap regions constitute large hydrophilic domains protruding from the extracellular membrane surface.

As stated above, the amino latch and the pre-stem regions suffer drastic conformational changes upon formation of the functional pore. The residues comprising the amino latch either in LukF (Olson et al. 1999) or in LukF-PV (Pedelac et al. 1999) adopt a P-strand conformation which forms part of the inner P-sheet of the sandwich domain. On the contrary, in the a-hemolysin protomer this region is irregular, establishing numerous contacts with one adjacent protomer (Fig. 3.13b). In the case of the pre-stem region, it forms a three-stranded antiparallel P-sheet packed against the P-sandwich, which in the oligomeric state extends away from it forming a definite P-hairpin. Noticeably, the region initially occupied by the pre-stem segment constitutes the binding site for the latch segment of the neighbour protomer.

Recently, the crystal structures of the water-soluble states of the eukaryotic PFPs equinatoxin II (Eqtll) from Actinia equina (Athanasiadis et al. 2001) and sticholysin II (Stnll) from Stichodactyla helianthus (Mancheno et al. 2003) (Fig. 3.11) have been reported. They are formed by a ten-stranded antiparallel P-sand-wich, which is flanked on each side by a short a-helix. Remarkably, whereas the longest connecting loops concentrate on one tip of the sandwich, the other tip is formed by tight turns. An important structural feature observed is the existence of a solvent-exposed region with a high concentration of aromatic residues, in which is located a phosphocholine-binding site (Fig. 3.11d) which may explain the high affinity these PFPs exhibit towards sphingomyelin (Mancheno et al. 2003). In this regard, recent mutagenesis studies on Stnll have shown the critical role of Tyr-111 in pore-formation (Alegre-Cebolleda et al. 2004), which perfectly agrees with the above structural studies. Additionally, electron microscopy (EM) analyses on 2D-crystals of Stnll formed on lipid monolayers (Martin-Benito et al.

2000; Mancheno et al. 2004) have shown the existence of tetrameric species with a pore-like topology which may represent a pre-pore state. Docking of the highresolution atomic model of Stnll into the EM envelope indicates the existence of conformational changes affecting the N-terminal helix, which may translocate essentially as a pseudo-rigid body from the P-sandwich to the membrane interface (Fig. 3.11B,e). This is in agreement with molecular biology results which showed that the presence of a disulfide bridge connecting the N-terminal helix and the P-sandwich inhibits pore-formation by Eqtll (Hong et al. 2003). Finally, a model for the functional pore has been proposed (Mancheno et al. 2003) based on the structure of the tetrameric species and previous extensive biochemical data (de los Rios et al. 1998; Anderluh and Macek 2002; Malovrh et al. 2003), in which these eukaryotic PFPs may form tetrameric toroidal pores with the N-terminal region adopting a helical conformation (Fig. 3.11c, f). The oligomeric assembly may interact with the membrane interface, with no protein segments spanning the hydrophobic core of the membrane.

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