Structure–Function Analysis of Individual Reversion Variants of the Engineered Mpp46Aa1 (PS2Aa1) N65 Protein

Background: Mpp46Aa1 (formerly PS2Aa1) is a β-pore-forming protein produced by Bacillus thuringiensis that exhibits selective cytotoxicity against cancer cells. Protein engineering has generated variants with improved antitumor activity, including the N65 variant, which contains three amino acid substitutions. Objective: In this study, three individual reversion variants of N65 were generated by site-directed mutagenesis to determine the contribution of each substitution to cytotoxicity, selectivity, and cell death mechanisms in the colorectal cancer cell lines SW480 and SW620 and the non-tumorigenic colonic epithelial cell line NCM460. Methods: Recombinant proteins were purified and evaluated using AlamarBlue viability assays, Annexin V/Cy3 staining, caspase-3/7 and caspase-9 activation assays, and JC-1 mitochondrial membrane potential analysis. Results: The reversion variants differentially affected the biological activity of N65. Among them, the D34N variant exhibited low IC50 values against both colorectal cancer cell lines, while maintaining selectivity towards non-cancerous cells. D34N also induced phosphatidylserine externalization, activation of caspase-3/7 and caspase-9, and mitochondrial membrane depolarization, indicating activation of the intrinsic apoptotic pathway. Structural analyses revealed that the reverted residues modified local interaction networks, suggesting that these positions may influence CD59 recognition and contribute to cellular selectivity. Conclusions: These findings demonstrate that the three substitutions present in N65 contribute unequally to its biological activity and identify residue 34 as a key determinant of Mpp46Aa1 cytotoxicity. This study provides structural and functional insights that support the rational engineering of parasporins with enhanced antitumor selectivity.