C-terminal amino acid residues are required for the folding and cholesterol binding property of perfringolysin O, a pore-forming cytolysin.
Perfringolysin O (theta-toxin) is a pore-forming cytolysin whose activity is triggered by binding to cholesterol in the plasma membrane. The cholesterol binding activity is predominantly localized in the beta-sheet-rich C-terminal half. In order to determine the roles of the C-terminal amino acids in theta-toxin conformation and activity, mutants were constructed by truncation of the C terminus. While the mutant with a two-amino acid C-terminal truncation retains full activity and has similar structural features to native theta-toxin, truncation of three amino acids causes a 40% decrease in hemolytic activity due to the reduction in cholesterol binding activity with a slight change in its higher order structure. Furthermore, both mutants were found to be poor at in vitro refolding after denaturation in 6 M guanidine hydrochloride, resulting in a dramatic reduction in cholesterol binding and hemolytic activities. These activity losses were accompanied by a slight decrease in beta-sheet content. A mutant toxin with a five-amino acid truncation expressed in Escherichia coli is recovered as a further truncated form lacking the C-terminal 21 amino residues. The product retains neither cholesterol binding nor hemolytic activities and shows a highly disordered structure as detected by alterations in the circular dichroism and tryptophan fluorescence spectra. These results show that the C-terminal region of theta-toxin has two distinct roles; the last 21 amino acids are involved to maintain an ordered overall structure, and in addition, the last two amino acids at the C-terminal end are needed for protein folding in vitro, in order to produce the necessary conformation for optimal cholesterol binding and hemolytic activities.
Shimada
Y
Y
Department of Protein Biochemistry, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan.
Nakamura
M
M
Naito
Y
Y
Nomura
K
K
Ohno-Iwashita
Y
Y
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000502:physiology*; D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D006460:Hemolysin Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006461:Hemolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D012756:Sheep; D013329:Structure-Activity Relationship
D000596:Amino Acids; D001427:Bacterial Toxins; D006460:Hemolysin Proteins; C020452:Clostridium perfringens theta-toxin; D002784:Cholesterol