Ribotoxins are a family of highly specific fungal
ribonucleases that inactivate the ribosomes by hydrolysis of a single phosphodiester bond of the 28 S rRNA.
alpha-Sarcin, the best characterized member of this family, is a potent
cytotoxin that promotes apoptosis of human
tumor cells after internalization via endocytosis. This latter ability is related to its interaction with
phospholipid bilayers. These
proteins share a common structural core with nontoxic
ribonucleases of the
RNase T1 family. However, significant structural differences between these two groups of
proteins are related to the presence of a long amino-terminal beta-hairpin in ribotoxins and to the different length of their unstructured loops. The amino-terminal deletion mutant Delta(7-22) of
alpha-sarcin has been produced in Escherichia coli and purified to homogeneity. It retains the same conformation as the wild-type
protein as ascertained by complete spectroscopic characterization based on circular dichroism, fluorescence, and NMR techniques. This mutant exhibits
ribonuclease activity against naked rRNA and synthetic substrates but lacks the specific ability of the wild-type
protein to degrade rRNA in intact ribosomes. The results indicate that
alpha-sarcin interacts with the ribosome at two regions, i.e. the well known sarcin-
ricin loop of the rRNA and a different region recognized by the beta-hairpin of the
protein. In addition, this latter
protein portion is involved in interaction with cell membranes. The mutant displays decreased interaction with
lipid vesicles and shows behavior compatible with the absence of one vesicle-interacting region. In agreement with this conclusion, the deletion mutant exhibits a very low cytotoxicity on human
rhabdomyosarcoma cells.