Three-Dimensional Structure of Different Functional Forms of the Vibrio cholerae Hemolysin Oligomer: a Cryo-Electron Microscopic Study

Vibrio cholerae hemolysin (HlyA) is a 65-kDa water-soluble pore-forming toxin that causes lysis of eukaryotic cells by destroying selective permeability of the plasma membrane bilayer. The HlyA monomer self-assembles on the target cell surface to the more stable β-barrel amphipathic heptamer, which inserts into the membrane bilayer to form a diffusion channel. Deletion of the 15-kDa β-prism lectin domain at the C terminus generates a 50-kDa hemolysin variant (HlyA50) with an ∼1,000-fold decrease in hemolytic activity. Because functional differences are eventually dictated by structural differences, we determined three-dimensional structures of 65- and 50-kDa HlyA oligomers, using cryo-electron microscopy and single-particle methods. Our study clearly shows that the HlyA oligomer has sevenfold symmetry but that the HlyA50 oligomer is an asymmetric molecule. The HlyA oligomer has bowl-like, arm-like, and ring-like domains. The bowl-like domain is coupled with the ring-like domain, and seven side openings are present just beneath the ring-like domain. Although a central channel is present in both HlyA and HlyA50 oligomers, they differ in pore size as well as in shape of the molecules and channel. These structural differences may be relevant to the striking difference in efficiencies of functional channel formation by the two toxin forms.Vibrio cholerae, a Gram-negative bacterium, is the causative agent of cholera in humans (9). The severe diarrhea of cholera is due primarily to the effects of cholera enterotoxin upon the small intestine epithelial cells (3). In addition to cholera toxin (9), most Vibrio cholerae strains secrete a membrane-damaging protein, designated cholera hemolysin (HlyA) (27) or cytolysin/hemolysin (VCC), with cytotoxic and cytolytic activity toward a wide spectrum of eukaryotic cells. VCC is an extracellular pore-forming toxin (PFT) (26) that exists in two stable forms: one is a water-soluble monomer with a molecular mass of 65 kDa (5, 6, 27, 31), and the other is a β-barrel amphipathic heptamer. The VCC monomer interacts with a cell surface receptor(s), self-assembles to an amphipathic β-barrel by circular oligomerization, and inserts into the membrane lipid bilayer (2, 7, 14, 21). Despite individual variations that depend on amino acid sequence, three-dimensional (3D) structure, and receptor specificity, PFTs have evolved a strikingly common strategy to destroy the permeability barrier of the target cell membrane (16). The soluble PFT monomer interacts with the target cell, self-assembles into an amphipathic β-barrel by circular oligomerization, and translocates from the cell surface or lipid-water interface to the core of the lipid bilayer, forming a transmembrane pore (5, 6).HlyA is expressed as an 82-kDa preprohemolysin protein, and following removal of the signal sequence, the protein is excreted during in vitro growth as the 79-kDa prohemolysin (proHlyA) (28). Proteolytic removal of 132 residues from the N-terminal region generates the mature 65-kDa HlyA, with a specific hemolytic activity of ∼100 pM toward rabbit erythrocytes (15). The mature toxin has a central cytolytic domain involved in oligomerization and anchoring to the hydrophobic core of the bilayer, followed by two lectin domains homologous to the carbohydrate-binding domains of the plant lectins ricin and jacalin (17). Proteolytic deletion of the 15-kDa β-prism lectin domain, which is apparently involved in specific interaction with β-galactosyl-terminated glycoconjugates (21), from the carboxy-terminal end of the 65-kDa mature hemolysin generates the truncated 50-kDa hemolysin (HlyA50) (8, 29). This truncated 50-kDa hemolysin (HlyA50) retains the oligomerization and membrane-anchoring domains (17, 18) and resembles the mature toxin in surface amphipathicity, nonspecific interaction with target biomembrane and lipid vesicles, and ability to undergo lipid-induced oligomerization (30). HlyA50 is about 1,000-fold less active than HlyA toward rabbit erythrocytes (29, 30), suggesting that the HlyA50 oligomer might be less capable of adopting an insertion-competent configuration.In the present study, we have attempted to determine the three-dimensional structures of the oligomers generated by the 65-kDa Vibrio cholerae hemolysin and the 50-kDa hemolysin, using cryo-electron microscopy and single-particle analysis techniques. Determination of the three-dimensional structure is important, as it might shed light on the shape of the transmembrane channel as well as on the anchoring and insertion processes of the two toxin forms.