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 residues). The three-dimensional structures of two of these proteins, alpha-sarcin and restrictocin, are known. They interact with phospholipid bilayers, according to their ability to enter cells, and cleave a specific phosphodiester bond in the large subunit of ribosome thus inhibiting protein biosynthesis. Two nonconservative sequence changes between these proteins are located 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 substituted with the equivalent amino acids in restrictocin. The single mutants (K11L and T20D) and the corresponding K11L/T20D double mutant have been produced in Escherichia coli and purified to homogeneity. The spectroscopic characterization of the purified proteins reveals that the overall native structure is preserved. The ribonuclease and lipid-perturbing activities of the three mutants and restrictocin have been evaluated and compared with those of alpha-sarcin. These proteins exhibit the same ability to specifically inactivate ribosomes, although they show different activity against nonspecific substrate analogs such as poly(A). The mutant variant K11L and restrictocin display a lower phospholipid-interacting ability correlated with a decreased cytotoxicity. The results obtained are interpreted in terms of the involvement of the amino-terminal beta-hairpin in the interaction with both membranes and polyadenylic acid.
García-Ortega
L
L
Departamento de Bioquímica y Biología Molecular, Facultad de Química, Universidad Complutense, 28040 Madrid, Spain.
Lacadena
J
J
Mancheño
J M
JM
Oñaderra
M
M
Kao
R
R
Davies
J
J
Olmo
N
N
Pozo AM
Gavilanes
J G
JG
D000485:Allergens; D019943:Amino Acid Substitution; D052179:Antigens, Plant; D001230:Aspergillus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003603:Cytotoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009183:Mycotoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000633:toxicity; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011500:Protein Synthesis Inhibitors / Q000737:chemistry / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D012270:Ribosomes / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014407:Tumor Cells, Cultured
C081947:ASPF1 protein, Aspergillus fumigatus; D000485:Allergens; D052179:Antigens, Plant; D003603:Cytotoxins; D005656:Fungal Proteins; D008051:Lipid Bilayers; D009183:Mycotoxins; D011500:Protein Synthesis Inhibitors; C014801:alpha-sarcin; C036196:MITF protein, Aspergillus restrictus; D004722:Endoribonucleases; D012260:Ribonucleases