Vibrio cholerae hemolysin. Implication of amphiphilicity and lipid-induced conformational change for its pore-forming activity.
Vibrio cholerae hemolysin (HlyA), a water-soluble protein with a native monomeric relative molecular mass of 65 000, forms transmembrane pentameric channels in target biomembranes. The HlyA binds to lipid vesicles nonspecifically and without saturation; however, self-assembly is triggered specifically by cholesterol. Here we show that the HlyA partitioned quantitatively to amphiphilic media irrespective of their compositions, indicating that the toxin had an amphiphilic surface. Asialofetuin, a beta1-galactosyl-terminated glycoprotein, which binds specifically to the HlyA in a lectin-glycoprotein type of interaction and inhibits carbohydrate-independent interaction of the toxin with lipid, reduced effective amphiphilicity of the toxin significantly. Resistance of the HlyA to proteases together with the tryptophan fluorescence emission spectrum suggested a compact structure for the toxin. Fluorescence energy transfer from the HlyA to dansyl-phosphatidylethanolamine required the presence of cholesterol in the lipid bilayer and was synchronous with oligomerization. Phospholipid bilayer without cholesterol caused a partial unfolding of the HlyA monomer as indicated by the transfer of tryptophan residues from the nonpolar core of the protein to a more polar region. These observations suggested: (a) partitioning of the HlyA to lipid vesicles is driven by the tendency of the amphiphilic toxin to reduce energetically unfavorable contacts with water and is not affected significantly by the composition of the vesicles; and (b) partial unfolding of the HlyA at the lipid-water interface precedes and promotes cholesterol-induced oligomerization to an insertion-competent configuration.
National Institute of Cholera and Enteric Diseases, Kolkata 700 010, India; Indian Institute of Chemical Biology, Kolkata 700 032, India.
Kausik
K
Chattopadhyay
Debasish
D
Bhattacharyya
Kalyan K
KK
Banerjee
D001426:Bacterial Proteins; D002784:Cholesterol / Q000494:pharmacology; D010450:Endopeptidases / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000737:chemistry*; D006460:Hemolysin Proteins / Q000737:chemistry*; D006461:Hemolysis; D006801:Humans; D007473:Ion Channels; D008051:Lipid Bilayers; D008081:Liposomes; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014734:Vibrio cholerae / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
D001426:Bacterial Proteins; D006460:Hemolysin Proteins; D007473:Ion Channels; D008051:Lipid Bilayers; D008081:Liposomes; C073845:hemolysin, Vibrio; D002784:Cholesterol; D010450:Endopeptidases