Background:Vibrio cholerae hemolysin is a pore-forming toxin with a ␤-prism lectin domain. Results: Although amphipathicity-driven interactions with lipids dominate membrane targeting, relocation of the lectin domain during toxin assembly generates entropy to push oligomerization and thereby promote pore formation. Conclusion:The lectin domain promotes toxin assembly by a carbohydrate-independent mechanism. Significance: A novel role for the lectin domain in protein oligomerization is revealed.

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 ␤-barrel amphipathic heptamer, which inserts into the membrane bilayer to form a diffusion channel. Deletion of the 15-kDa ␤-prism lectin domain at the C terminus generates a 50-kDa hemolysin variant (HlyA50) with an ϳ1,000-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.

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