Minimized natural versions of fungal ribotoxins show improved active site plasticity

Abstract

12Fungal ribotoxins are highly specific extracellular RNases which cleave a single [23, 24, 32, 35]. Therefore, they can be considered as minimized natural versions 57 of their larger counterparts. This lower sequence identity and size do not however preclude 58 the conservation of the elements of ordered secondary structure as well as the identity and 59 geometric arrangement of the residues configuring the active site (Figure 1). This structural 60 conservation can be even extended to the other non-toxic members of the larger fungal 61 extracellular RNases family, such as RNase T1 and RNase U2, for example [36, 37]. In 74In the work herein presented α-sarcin Tyr 48 has been replaced by an Asp residue to 75 produce and characterize the corresponding Y48D mutant. As a control, the corresponding 76 inverse anisoplin mutant (D43Y) has been also studied. The results shown reveal the key 77 role of these residues not only in maintaining the correct electrostatic environment and 78 active site plasticity in each type of ribotoxins but also in the preservation of their 79 characteristic high thermostability. 80 MATERIALS AND METHODS 81 Mutant cDNA construction 82All materials and reagents were of molecular biology grade. Cloning procedures, PCR-83 based oligonucleotide site-directed mutagenesis, and bacterial manipulations were carried 84 out as previously described [11, 16,[41][42][43]. Mutagenesis constructions were performed 85 using different sets of complementary mutagenic primers (Table S1). Mutations were 86 confirmed by DNA sequencing at the corresponding Complutense University facility. The 87 plasmids used as templates for mutagenesis, containing the cDNA sequence of either wild-88 type α-sarcin or anisoplin, have already been described [35, 38, 42]. 89 Protein production and purification 90Escherichia coli RB791 or BL21 (DE3) cells, the latter ones being previously 91 cotransformed with a thioredoxin-producing plasmid (pT-Trx), and the corresponding wild-92 type or mutant plasmids were used to produce and purify all proteins from the periplasmic 93 soluble fraction, as previously described [35, 38, 42,[44][45][46]. The only exception was fungal 94 wild-type α-sarcin which was isolated from Aspergillus giganteus MDH18894, its natural 95 source, following the procedure reported before [11]. SDS-PAGE of proteins, Western 96 blots, protein hydrolysis, and amino acid analysis were performed according to standard 97 procedures, also as previously described [11, 42]. All four proteins studied were purified to 98 homogeneity according to their SDS-PAGE behavior and amino acid analysis. 99 Spectroscopic characterization 100Spectroscopic characterization was performed following well established procedures [11, 101 22, 38, 41, 44, 47-51]. Absorbance measurements were carried out on a Shimadzu UV- Ribonucleolytic activity assays 118The ribonucleolytic activity of ribotoxins on rabbit ribosomes was followed by detecting 119 the release of a specific 400-nt rRNA fragment, known as the α-fragment, from ...