Involvement of the amino-terminal beta-hairpin of the Aspergillus ribotoxins on the interaction with membranes and nonspecific ribonuclease activity

Ribotoxins are a family of potent cytotoxic proteins from Aspergillus whose members display a high sequence identity (85% for about 150 amino acid res idues). The three-dimensional structures of two of these proteins, alpha -s arcin and restrictocin, are known. They interact with phospholipid bilayers , according to their ability to enter cells, and cleave a specific phosphod iester bond in the large subunit of ribosome thus inhibiting protein biosyn thesis. Two nonconservative sequence changes between these proteins are loc ated at the amino-terminal beta -hairpin of alpha -sarcin, a characteristic structure that is absent in other nontoxic structurally related microbial RNases. These two residues of alpha -sarcin, Lys 11 and Thr 20, have been s ubstituted with the equivalent amino acids in restrictocin, The single muta nts (K11L and T20D) and the corresponding K11L/T20D double mutant have been produced in Escherichia coli and purified to homogeneity. The spectroscopi c characterization of the purified proteins reveals that the overall native structure is preserved. The ribonuclease and lipid-perturbing activities o f the three mutants and restrictocin have been evaluated and compared with those of alpha -sarcin. These proteins exhibit the same ability to specific ally inactivate ribosomes, although they show different activity against no nspecific substrate analogs such as poly(A). The mutant variant Kill, and r estrictocin display a lower phospholipid-interacting ability correlated wit h a decreased cytotoxicity. The results obtained are interpreted in terms o f the involvement of the amino-terminal beta -hairpin in the interaction wi th both membranes and polyadenylic acid.