Deletion of the NH2-terminal beta-hairpin of the ribotoxin alpha-sarcin produces a nontoxic but active ribonuclease.
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.
Garcia-Ortega
Lucia
L
Departamento de Bioquimica y Biologia Molecular I, Universidad Complutense, Madrid 28040, Spain.
Masip
Manuel
M
Mancheño
José M
JM
Oñaderra
Mercedes
M
Lizarbe
M Antonia
MA
García-Mayoral
M Flor
MF
Bruix
Marta
M
Martínez del Pozo
Alvaro
A
Gavilanes
José G
JG
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011489:Protein Denaturation; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013057:Spectrum Analysis
D017931:DNA Primers; D005656:Fungal Proteins; C014801:alpha-sarcin; D004722:Endoribonucleases; C022544:ribonuclease U2