Álvarez García, Elisa and Martínez del Pozo, Álvaro and Gavilanes, José G. (2009) Role of the basic character of α-sarcin’s NH2-terminal β-hairpin in ribosome recognition and phospholipid interaction. Archives of Biochemistry and Biophysics, 481 . pp. 37-44. ISSN 0003-9861
Ribotoxins are a family of toxic extracellular fungal RNases that first enter into the cells and then exert a highly specific ribonucleolytic activity on the larger rRNA molecule, leading to protein synthesis inhibition and cell death by apoptosis. α-Sarcin is the best characterized ribotoxin. Previous characterization of a deletion variant of this protein showed that its long NH2-terminal β-hairpin is essential for its cytotoxicity. Docking, enzymatic, and lipid-protein interaction studies suggested that this β-hairpin establishes specific interactions with ribosomal proteins and that it is a region involved in the interaction with cell membranes. Consequently, in order to assess the influence of the basic character of this NH2-terminal β-hairpin (there are 1 arginine and 4 lysines along its 16 residues) on the ribotoxins cytotoxic ability, five individual mutants substituting these 5 basic residues by glutamic acid were produced, purified to homogeneity, and characterized. Regarding ribosomal recognition, all mutants showed a diminished activity in a cell-free reticulocyte lysate, whereas the activity against an oligoribonucleotide mimicking the sarcin/ricin loop rRNA (SRL) or the homopolymer poly(A) remained unaffected, confirming that the mutated basic residues participate in electrostatic interactions with other ribosomal elements apart from this SRL. The study of the interaction with phospholipid vesicles showed that Lys 17, Arg 22, and, most importantly, Lys 14 and Lys 21, are crucial residues in the first stages of the aggregation phenomenon, where protein-vesicle and protein-protein interactions are required. The data obtained reveal that electrostatic interactions involving basic residues of the β-hairpin are required not only for establishing specific interactions with ribosomal regions other than the SRL but also to explain the ability of the protein to interact with acid phospholipid bilayers.
|Uncontrolled Keywords:||Ribotoxin, Protein-lipid interaction, Ribosome recognition, Restrictocin, Electrostatic interaction, Ribonuclease, Site-directed mutagenesis|
|Subjects:||Sciences > Chemistry > Biochemistry|
|Deposited On:||08 Jun 2009 16:30|
|Last Modified:||07 Sep 2010 10:30|
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