Role of the basic character of α-sarcin’s NH2-terminal β-hairpin in ribosome recognition and phospholipid interaction

Abstract

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 NH 2 -terminal β-hairpin is essential for its cytotoxicity. Docking, enzymatic, and lipidprotein 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 NH 2 -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.

Ribonucleases as a host-defence family: evidence of evolutionarily conserved antimicrobial activity at the N-terminus

“…The evolutionary distances were computed using the Poisson distribution method [40] and are in units of number of amino acid substitutions per site. Interestingly, we have also found comparable behaviours in other cytotoxic RNase families, such as the contribution of the N-terminus of ribotoxin in the interaction with lipid bilayers [34]. Evolutionary analyses were conducted in MEGA5 [41].…”

Interaction of Nuclease Colicins with Membranes: Insertion Depth Correlates with Bilayer Perturbation

“…Alpha-sarcin contains three discrete membrane interaction regions, one of which, the N-terminal beta hairpin, is not only required to target specific ribosomal proteins, but is also the region where many of the electrostatic interactions with the acidic membrane surface take place [67]. It is tempting to speculate that the N-terminal immunity protein binding site of the rRNase E3 which is also involved in ribosomal protein binding [68], is equally important in the membrane targeting process, given its basic nature, amphipathic character, inherent flexibility, lack of interactions with the main beta sheet and absence of membrane binding in the presence of immunity protein.…”

Involvement of loop 5 lysine residues and the N-terminal β-hairpin of the ribotoxin hirsutellin A on its insecticidal activity

“…Spectroscopic characterization was performed following well-established procedures (Álvarez-García et al 2006;Álvarez-García et al 2009b;García-Ortega et al 2005a;García-Ortega et al 2001;García-Ortega et al 2002;Lacadena et al 1999;Martínez-Ruiz et al 2001). Absorbance measurements were carried out on a Beckman DU640 were also used to calculate their extinction coefficients (Table 1).…”

“…In fact, it has been reported before how α-sarcin Lys 14 and 21, with identical amino acids in equivalent positions of HtA (Fig. 1), are crucial residues for the correct achievement of these interactions (Álvarez-García et al 2009b). …”