title: Implication of an Asp residue in the ribonucleolytic activity of hirsutellin A reveals new electrostatic interactions at the active site of ribotoxins.
abstract: Ribotoxins are fungal extracellular ribonucleases that specifically cleave ribosomes leading to cell-death via apoptosis. α-Sarcin is the ribotoxin studied in deepest detail, and therefore constitutes the referential protein for the whole family. It has been demonstrated that ribotoxin activity depends on a very precise structural microenvironment in which electrostatic interactions among residues in the active site are of the highest importance. Hirsutellin A (HtA) has been recently described as the smallest ribotoxin known to date, encompassing all the abilities of previously characterized members of this family into a shorter sequence. Comparison of HtA and α-sarcin three-dimensional structures suggested that residues presumably forming the catalytic triad of HtA would be His 42, Glu 66, and His 113. Within this same idea, the presence of an Asp residue (Asp 40) in a position equivalent to α-sarcin Tyr 48 is highlighted as a novelty in this field. In this work, substitution mutants H42Q, E66Q and H113Q, as well as double and triple mutants in all possible combinations, are studied regarding their ribonucleolytic activity and cytotoxicity. Implication of these three residues in the ribotoxin activity of HtA is confirmed, though none of them is strictly essential for ribosomal cleavage. Studies with mutants D40N and D40N/E66Q demonstrate an important role for Asp 40 in the activity of HtA and establish a new set of electrostatic interactions different from the one described for already known ribotoxins.
authors.affiliation: Departamento de Bioquímica y Biología Molecular I, Facultad de Química, Universidad Complutense, 28040 Madrid, Spain.
authors.forename: Elías
authors.initials: E
authors.lastname: Herrero-Galán
authors.forename: Lucía
authors.initials: L
authors.lastname: García-Ortega
authors.forename: Javier
authors.initials: J
authors.lastname: Lacadena
authors.forename: Alvaro
authors.initials: A
authors.lastname: Martínez-Del-Pozo
authors.forename: Nieves
authors.initials: N
authors.lastname: Olmo
authors.forename: José G
authors.initials: JG
authors.lastname: Gavilanes
authors.forename: Mercedes
authors.initials: M
authors.lastname: Oñaderra
mesh_terms: D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D014176:Protein Biosynthesis / Q000187:drug effects*; D011487:Protein Conformation; D011500:Protein Synthesis Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D012270:Ribosomes / Q000378:metabolism*; D055672:Static Electricity
chemical_list: D005656:Fungal Proteins; C121692:Hirsutellin A protein, Hirsutella thompsonii; D011500:Protein Synthesis Inhibitors; D011994:Recombinant Proteins; C014801:alpha-sarcin; D001224:Aspartic Acid; D004722:Endoribonucleases