title: The β-prism lectin domain of Vibrio cholerae hemolysin promotes self-assembly of the β-pore-forming toxin by a carbohydrate-independent mechanism.
abstract: Vibrio cholerae cytolysin/hemolysin (VCC) is an amphipathic 65-kDa β-pore-forming toxin with a C-terminal β-prism lectin domain. Because deletion or point mutation of the lectin domain seriously compromises hemolytic activity, it is thought that carbohydrate-dependent interactions play a critical role in membrane targeting of VCC. To delineate the contributions of the cytolysin and lectin domains in pore formation, we used wild-type VCC, 50-kDa VCC (VCC(50)) without the lectin domain, and mutant VCC(D617A) with no carbohydrate-binding activity. VCC and its two variants with no carbohydrate-binding activity moved to the erythrocyte stroma with apparent association constants on the order of 10(7) M(-1). However, loss of the lectin domain severely reduced the efficiency of self-association of the VCC monomer with the β-barrel heptamer in the synthetic lipid bilayer from ∼83 to 27%. Notably, inactivation of the carbohydrate-binding activity by the D617A mutation marginally reduced oligomerization to ∼77%. Oligomerization of VCC(50) was temperature-insensitive; by contrast, VCC self-assembly increased with increasing temperature, suggesting that the process is driven by entropy and opposed by enthalpy. Asialofetuin, the β1-galactosyl-terminated glycoprotein inhibitor of VCC-induced hemolysis, promoted oligomerization of 65-kDa VCC to a species that resembled the membrane-inserted heptamer in stoichiometry and morphology but had reduced global amphipathicity. In conclusion, we propose (i) that the β-prism lectin domain facilitated toxin assembly by producing entropy during relocation in the heptamer and (ii) that glycoconjugates inhibited VCC by promoting its assembly to a water-soluble, less amphipathic oligomer variant with reduced ability to penetrate the bilayer.
authors.affiliation: From the Divisions of Biochemistry and.
authors.forename: Sreerupa
authors.initials: S
authors.lastname: Ganguly
authors.forename: Amarshi
authors.initials: A
authors.lastname: Mukherjee
authors.forename: Budhaditya
authors.initials: B
authors.lastname: Mazumdar
authors.forename: Amar N
authors.initials: AN
authors.lastname: Ghosh
authors.forename: Kalyan K
authors.initials: KK
authors.lastname: Banerjee
mesh_terms: D019943:Amino Acid Substitution; D001212:Asialoglycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D060748:Fetuins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism; D020125:Mutation, Missense; D055503:Protein Multimerization / Q000502:physiology*; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D014734:Vibrio cholerae / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001212:Asialoglycoproteins; D001426:Bacterial Proteins; D060748:Fetuins; D006460:Hemolysin Proteins; D008051:Lipid Bilayers; C017029:asialofetuin; C073845:hemolysin, Vibrio
keywords: Carbohydrate Glycoprotein; Lectin; Lipid-binding Protein; Liposomes; Pore-forming Toxins; Protein Assembly; Vibrio cholerae Hemolysin