Involvement of loops 2 and 3 of α-sarcin on its ribotoxic activity

•The role of loops 2 and 3 in the toxic activity of ribotoxins has been only poorly studied.•Results presented now confirm the key role of loop 3 Lys-111, 112, and 114 for the SRL specific recognition.•They also show the existence of a network of interactions involving Lys-114 and Tyr-48 which is essential for catalysis.•Loop 3 Lys-rich region is involved in electrostatic interactions needed to cross cell membranes.•Loop 2 loop is responsible for easing the onset of hydrophobic interactions with the membrane inner leaflets.

Ribotoxins are a family of fungal ribosome-inactivating proteins displaying highly specific ribonucleolytic activity against the sarcin/ricin loop (SRL) of the larger rRNA, with α-sarcin as its best-characterized member. Their toxicity arises from the combination of this activity with their ability to cross cell membranes. The involvement of α-sarcin's loops 2 and 3 in SRL and ribosomal proteins recognition, as well as in the ribotoxin-lipid interactions involving cell penetration, has been suggested some time ago. In the work presented now different mutants have been prepared in order to study the role of these loops in their ribonucleolytic and lipid-interacting properties. The results obtained confirm that loop 3 residues Lys 111, 112, and 114 are key actors of the specific recognition of the SRL. In addition, it is also shown that Lys 114 and Tyr 48 conform a network of interactions which is essential for the catalysis. Lipid-interaction studies show that this Lys-rich region is indeed involved in the phospholipids recognition needed to cross cell membranes. Loop 2 is shown to be responsible for the conformational change which exposes the region establishing hydrophobic interactions with the membrane inner leaflets and eases penetration of ribotoxins target cells.

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