The C-terminal peptide produced upon proteolytic activation of the cytolytic toxin aerolysin is not involved in channel formation.
The channel-forming toxin aerolysin is secreted by Aeromonas hydrophila as a protoxin that can be activated by nicking with endoproteinase Lys-C after Lys-427 near the C terminus of the protein. The fate of the 43-amino acid peptide distal to the activation site was investigated. A cysteine was introduced into the C-terminal region by replacing Ile-445, and another replaced Gly-202, which is on the proximal side of the activation site. In a double mutant, the two new cysteines were close enough in the folded molecule to form an intrachain 202-445 disulfide bond. Tryptophan fluorescence measurements on wild type and the 2 single cysteine mutants indicated that activation results in exposure of at least 1 tryptophan residue, leading to the conclusion that the peptide moves with respect to the protein when it is produced. This was supported by the observation that upon activation there was a decrease in energy transfer between a tryptophan in the bulk of the protein and a probe attached to Cys-445. The peptide could be separated from active toxin by several methods, indicating that it leaves the protein when it is produced, and that it plays no further role in the process of channel formation.
van der Goot
F G
FG
Département de Biochimie, Université de Genève, Switzerland.
Hardie
K R
KR
Parker
M W
MW
Buckley
J T
JT
D016980:Aeromonas hydrophila / Q000378:metabolism*; D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D002942:Circular Dichroism; D003545:Cysteine; D010450:Endopeptidases / Q000378:metabolism; D005456:Fluorescent Dyes; D007473:Ion Channels / Q000737:chemistry; D007532:Isoleucine; D008239:Lysine; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D052899:Pore Forming Cytotoxic Proteins; D011487:Protein Conformation; D011498:Protein Precursors / Q000737:chemistry* / Q000378:metabolism; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000032:analysis
D001427:Bacterial Toxins; D005456:Fluorescent Dyes; D007473:Ion Channels; D010446:Peptide Fragments; D052899:Pore Forming Cytotoxic Proteins; D011498:Protein Precursors; D011994:Recombinant Proteins; D007532:Isoleucine; C032676:aerolysin; D014364:Tryptophan; D010450:Endopeptidases; D008239:Lysine; D003545:Cysteine