Trp132, Trp154, and Trp157 are essential for folding and activity of a Cyt toxin from Bacillus thuringiensis

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Bacillus thuringiensis subsp. darmstadiensis produces the Cyt2Aa2 toxin, known for its cytolytic and mosquito larvicidal properties.
• The Cyt2Aa2 toxin possesses three critical tryptophan residues at positions 132, 154, and 157, suggesting their importance in protein function.

Purpose of the Study:

• To elucidate the specific roles of tryptophan residues in the structure and function of the Cyt2Aa2 toxin.
• To determine the impact of substituting these tryptophan residues with other amino acids on the toxin's haemolytic and larvicidal activities.

Main Methods:

• Site-directed mutagenesis was employed to substitute tryptophan residues at positions 132, 154, and 157 with phenylalanine and other amino acids.
• Mutant proteins were expressed in Escherichia coli, and their production as inclusion bodies was assessed.
• Haemolytic and mosquito larvicidal activities of the wild-type and mutant toxins were evaluated.
• Solubilization and proteinase K activation assays were performed to analyze protein stability and activation.

Main Results:

• All expressed mutant proteins formed inclusion bodies, similar to the wild-type Cyt2Aa2 toxin.
• The W157F mutant retained significant haemolytic and mosquito larvicidal activities, comparable to the wild type.
• Mutations at tryptophan positions 132 and 154, and the W157V mutation, resulted in a complete loss of both haemolytic and larvicidal activities.
• Analysis indicated that an aromatic residue is essential at position 157, while tryptophan is critical at positions 132 and 154 for maintaining Cyt2Aa2's structure and function.

Conclusions:

• Tryptophan residues at positions 132 and 154 are indispensable for the structural integrity and functional activity of the Cyt2Aa2 toxin.
• Position 157 requires an aromatic residue for activity, but allows for substitution beyond tryptophan.
• These findings highlight the critical role of specific amino acid residues in the function of microbial toxins and provide insights for protein engineering efforts.