Crystal structure of binase does not reflect its native conformation

Cytotoxic ribonucleases (RNases) of the T1 family, including binase, the secreted guanyl-preferring RNase of Bacillus pumilus, are considered as promising agents of alternative anticancer chemotherapy. Binase has a selective apoptosis-inducing action against cells expressing oncogenes ras, kit, AML-ETO. The crystal structure of the binase mutant with two-point amino acid substitutions at positions 43 and 81 (Glu43Ala/Phe81Ala) indicates the absence of dimeric forms, which are characteristic for the wild-type binase. We studied the native structural organization of the Glu43Ala/Phe81Ala mutant. It was found that the mutant enzyme, similarly to the wild-type binase, possesses catalytically active dimers of different stability levels identified not only under denaturing gel electrophoresis, but also under native conditions. The results of the study show that binase exists predominantly in the dimeric form, whereas the Glu43Ala/Phe81Ala mutant approximately equally represented by both dimers and monomers formed as a result of the decomposition of unstable dimers. Although the catalytic activity of the mutant with respect to the natural substrate, RNA, was lower, as compared to the wild-type enzyme, it exhibited a 32–35% higher cytotoxicity against human adenocarcinoma cells. The data obtained indicate the contribution of the structural organization of RNases to their cytotoxicity and confirm the significance of the analysis of the native conformation of cytotoxic proteins