Unusual proteolysis of the protoxin and toxin from Bacillus thuringiensis. Structural implications.
Trypsin is shown to generate an insecticidal toxin from the 130-kDa protoxin of Bacillus thuringiensis subsp. kurstaki HD-73 by an unusual proteolytic process. Seven specific cleavages are shown to occur in an ordered sequence starting at the C-terminus of the protoxin and proceeding toward the N-terminal region. At each step, C-terminal fragments of approximately 10 kDa are produced and rapidly proteolyzed to small peptides. The sequential proteolysis ends with a 67-kDa toxin which is resistant to further proteolysis. However, the toxin could be specifically split into two fragments by proteinases as it unfolded under denaturing conditions. Papain cleaved the toxin at glycine 327 to give a 34.5-kDa N-terminal fragment and a 32.3-kDa C-terminal fragment. Similar fragments could be generated by elastase and trypsin. The N-terminal fragment corresponds to the conserved N-terminal domain predicted from the gene-deduced sequence analysis of toxins from various subspecies of B. thuringiensis, and the C-terminal fragment is the predicted hypervariable sequence domain. A double-peaked transition was observed for the toxin by differential scanning calorimetry, consistent with two or more independent folding domains. It is concluded that the N- and C-terminal regions of the protoxin are two multidomain regions which give unique structural and biological properties to the molecule.
Department of Chemistry, University of Ottawa, Canada.
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Choma
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Surewicz
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Carey
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Pozsgay
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Raynor
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Kaplan
D000595:Amino Acid Sequence; D001413:Bacillus thuringiensis / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D001427:Bacterial Toxins / Q000378:metabolism*; D001665:Binding Sites; D002152:Calorimetry, Differential Scanning; D007306:Insecticides / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D010447:Peptide Hydrolases; D011498:Protein Precursors / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013696:Temperature
C029103:Bacillus thuringiensis protoxin; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D007306:Insecticides; D010446:Peptide Fragments; D011498:Protein Precursors; D010447:Peptide Hydrolases