Leucine 145 of the ribotoxin alpha-sarcin plays a key role for determining the specificity of the ribosome-inactivating activity of the protein

Secreted fungal RNases, represented by RNase T1, constitute a family of structurally related proteins that includes ribotoxins such as alpha-sarcin. The active site residues of RNase T1 are conserved in all fungal RNases, except for Phe 100 that is not present in the ribotoxins, in which Leu 145 occupies the equivalent position. The mutant Leu145Phe of alpha-sarcin has been recombinantly produced and characterized by spectroscopic methods (circular dichroism, fluorescence spectroscopy, and NMR). These analyses have revealed that the mutant protein retained the overall conformation of the wild-type alpha-sarcin. According to the analyses performed, Leu 145 was shown to be essential to preserve the electrostatic environment of the active site that is required to maintain the anomalous low pKa value reported for the catalytic His 137 of alpha-sarcin. Enzymatic characterization of the mutant protein has revealed that Leu 145 is crucial for the specific activity of alpha-sarcin on ribosomes.     

Deletion of the NH2-terminal beta-hairpin of the ribotoxin alpha-sarcin produces a nontoxic but active ribonuclease

Ribotoxins are a family of highly specific fungal ribonucleases that inactivate the ribosomes by hydrolysis of a single phosphodiester bond of the 28 S rRNA. alpha-Sarcin, the best characterized member of this family, is a potent cytotoxin that promotes apoptosis of human tumor cells after internalization via endocytosis. This latter ability is related to its interaction with phospholipid bilayers. These proteins share a common structural core with nontoxic ribonucleases of the RNase T1 family. However, significant structural differences between these two groups of proteins are related to the presence of a long amino-terminal beta-hairpin in ribotoxins and to the different length of their unstructured loops. The amino-terminal deletion mutant Delta(7-22) of alpha-sarcin has been produced in Escherichia coli and purified to homogeneity. It retains the same conformation as the wild-type protein as ascertained by complete spectroscopic characterization based on circular dichroism, fluorescence, and NMR techniques. This mutant exhibits ribonuclease activity against naked rRNA and synthetic substrates but lacks the specific ability of the wild-type protein to degrade rRNA in intact ribosomes. The results indicate that alpha-sarcin interacts with the ribosome at two regions, i.e. the well known sarcin-ricin loop of the rRNA and a different region recognized by the beta-hairpin of the protein. In addition, this latter protein portion is involved in interaction with cell membranes. The mutant displays decreased interaction with lipid vesicles and shows behavior compatible with the absence of one vesicle-interacting region. In agreement with this conclusion, the deletion mutant exhibits a very low cytotoxicity on human rhabdomyosarcoma cells.     

Arginine 121 is a crucial residue for the specific cytotoxic activity of the ribotoxin alpha-sarcin.

Alpha-sarcin, a cyclizing ribonuclease secreted by the mould Aspergillus giganteus, is one of the best characterized members of a family of fungal ribotoxins. This protein induces apoptosis in tumour cells due to its highly specific activity on ribosomes. Fungal ribotoxins display a three-dimensional protein fold similar to those of a larger group of microbial noncytotoxic RNases, represented by RNases T1 and U2. This similarity involves the three catalytic residues and also the Arg121 residue, whose counterpart in RNase T1, Arg77, is located in the vicinity of the substrate phosphate moiety although its potential functional role is not known. In this work, Arg121 of alpha-sarcin has been replaced by Gln or Lys. These two mutations do not modify the conformation of the protein but abolish the ribosome-inactivating activity of alpha-sarcin. In addition, the loss of the positive charge at that position produces dramatic changes on the interaction of alpha-sarcin with phospholipid membranes. It is concluded that Arg121 is a crucial residue for the characteristic cytotoxicity of alpha-sarcin and presumably of the other fungal ribotoxins.     

Tyr-48, a conserved residue in ribotoxins, is involved in the RNA-degrading activity of alpha-sarcin

Residue Tyr-48 in alpha-sarcin is conserved not only within the ribotoxin family, but also within the larger group of extracellular fungal ribonucleases, best represented by RNase T1. A mutant protein in which this Tyr residue was substituted by Phe has been produced and isolated to homogeneity. It was spectroscopically analyzed by means of circular dichroism, fluorescence emission and NMR. Taken together, these results and those from enzyme characterization have revealed the essential role of the -OH group from the Tyr-48 phenolic ring in the cleavage of polymeric RNA substrates, including the ribosome-embedded 28S rRNA, the natural substrate of ribotoxins. Thus, the mutant protein does not degrade its natural ribosomal RNA substrate. However, it has been shown that this Y48F mutant still retains its ability to cleave a phosphodiester bond in a minimal substrate such as the dinucleoside phosphate ApA. The role of different alpha-sarcin residues within the enzyme reaction catalyzed by this protein is discussed.     

Tautomeric state of alpha-sarcin histidines. Ndelta tautomers are a common feature in the active site of extracellular microbial ribonucleases

Extracellular fungal RNases, including ribotoxins such as alpha-sarcin, constitute a family of structurally related proteins represented by RNase T1. The tautomeric preferences of the alpha-sarcin imidazole side chains have been determined by nuclear magnetic resonance and electrostatic calculations. Histidine residues at the active site, H50 and H137, adopt the Ndelta tautomer, which is less common in short peptides, as has been found for RNase T1. Comparison with tautomers predicted from crystal structures of other ribonucleases suggests that two active site histidine residues with the Ndelta tautomer are a conserved feature of microbial ribonucleases and that this is related to their ribonucleolytic function.     

Molecular dissection of mitogillin reveals that the fungal ribotoxins are a family of natural genetically engineered ribonucleases

Mitogillin and the related fungal ribotoxins are highly specific ribonucleases which inactivate the ribosome enzymatically by cleaving the 23-28 S RNA of the large ribosomal subunit at a single phosphodiester bond. The site of cleavage occurs between G4325 and A4326 (rat ribosome numbering) which are present in one of the most conserved sequences (the alpha-sarcin loop) among the large subunit ribosomal RNAs of all living species. Amino acid sequence comparison of ribotoxins and guanyl/purine ribonucleases have identified domains or residues likely involved in ribonucleolytic activity or cleavage specificity. Fifteen deletion mutants (each 4 to 8 amino acid deletions) in motifs of mitogillin showing little amino acid sequence homology with guanyl/purine ribonucleases were constructed by site-directed mutagenesis. Analyses of the purified mutant proteins identified those regions in fungal ribotoxins contributing to ribosome targeting and modulating the catalytic activity of the toxin; some of the identified motifs are homologous to sequences in ribosomal proteins and elongation factors. This mutational study of mitogillin together with the recently published x-ray structure of restrictocin (a close relative of mitogillin) supports the hypothesis that the specific cleavage properties of ribotoxins are the result of natural genetic engineering in which the ribosomal targeting elements of ribosome-associated proteins were inserted into nonessential regions of T1-like ribonucleases.     

Conserved asparagine residue 54 of alpha-sarcin plays a role in protein stability and enzyme activity

Asparagine 54 of alpha-sarcin is a conserved residue within the proteins of the ribotoxin family of microbial ribonucleases. It is located in loop 2 of the protein, which lacks repetitive secondary structure elements but exhibits a well-defined conformation. Five mutant variants at this residue have been produced and characterized. The spectroscopic characterization of these proteins indicates that the overall conformation is not changed upon mutation. Activity and denaturation assays show that Asn-54 largely contributes to protein stability, and its presence is a requirement for the highly specific inhibitory activity of these ribotoxins on ribosomes.     

Modeling the highly specific ribotoxin recognition of ribosomes

The three-dimensional structures of the alpha-sarcin ribotoxin and its delta(7-22) deletion mutant, both complexed with a 20-mer oligonucleotide mimicking the sarcin/ricin loop (SRL) of the ribosome, have been docked into the structure of the Halobacterium marismortui ribosome by fitting the nucleotide atomic coordinates into those of the ribosomal SRL. This study has revealed that two regions of the ribotoxin, residues 11-16 and 84-85, contact the ribosomal proteins L14 (residues 99-105) and L6 (residues 88-92), respectively. The first of these two ribotoxin regions appears to be crucial for its specific ribosome recognition.     

Domain swapping in ribonuclease T1 allows the acquisition of double-stranded activity

A mutant of ribonuclease T1 (RNase T1), denoted RNase Talpha, that is designed to recognize double-stranded ribonucleic acid was created. RNase Talpha carries the structure of RNase T1 except for a part of its loop L3 domain, which has been swapped for a corresponding domain from alpha-sarcin. The RNase Talpha maintains the pleated beta-sheet structure and retains the guanyl-specific ribonuclease activity of the wild-type RNase T1. A steady-state kinetic study on the RNase Talpha-catalyzed transesterification of GpU dinucleoside phosphates reveals a slightly reduced K(m) value of 6.94 x 10(-7) M. When the stranded specificity is examined, RNase Talpha catalyzes the hydrolysis of guanine base not only of single-stranded but also, as by design, of double-stranded RNA. The change of stranded specificity suggests the feasibility of using domain swapping to make a substrate-specific ribonuclease. This study suggests that the loop L3 in RNase T1 can be used as a 'cassette player' for inserting a functional domain to make ribonuclease of various specificities.     

Effects of antisense DNA against the alpha-sarcin stem-loop structure of the ribosomal 23S rRNA.

Antisense DNAs complementary against various sequences of the alpha-sarcin domain (C2646-G2674) of 23S rRNA from Escherichia coli were hybridized to naked 23S rRNA as well as to 70S ribosomes. Saturation levels of up to 0.4 per 70S ribosome were found, the identical fraction was susceptible to the attack of the RNase alpha-sarcin. The hybridization was specific as demonstrated with RNase H digestion, sequencing the resulting fragments and blockage of the action of alpha-sarcin. The RNase alpha-sarcin seems to approach its cleavage site from the 3' half of the loop of the alpha-sarcin domain. Hybridization is efficiently achieved at 37 degrees C and can extend at least into the 3' strand of the stem of the alpha-sarcin domain. However, the inhibition of alpha-sarcin activity is observed at 30 degrees C but not at 37 degrees C. For a significant inhibition of poly(Phe) synthesis the temperature had to be lowered to 25 degrees C. The results imply that the alpha-sarcin domain changes its conformation during protein synthesis and that the conformational changes may include a melting of the stem of the alpha-sarcin domain.     

The action mode of the ribosome-inactivating protein α-sarcin

Based on the tertiary structure of the ribosome-inactivating protein alpha-sarcin, domains that are responsible for hydrolyzing ribosomes and naked RNA have been dissected. In this study, we found that the head-to-tail interaction between the first amino beta-strand and the last carboxyl beta-strand is not involved in catalyzing the hydrolysis of ribosomes or ribonucleic acids. Instead, a four-strand pleated beta-sheet is indispensable for catalyzing both substrates, suggesting that alpha-sarcin and ribonuclease T1 (RNase T1) share a similar catalytic center. The integrity of an amino beta-hairpin and that of the loop L3 in alpha-sarcin are crucial for recognizing and hydrolyzing ribosomes in vitro and in vivo. However, a mutant protein without the beta-hairpin structure, or with a disrupted loop L3, is still capable of digesting ribonucleic acids. The functional involvement of the beta-hairpin and the loop L3 in the sarcin stem/loop RNA of ribosomes is demonstrated by a docking model, suggesting that the two structures are in essence naturally designed to distinguish ribosome-inactivating proteins from RNase T1 to inactivate ribosomes.     

Production and characterization of a noncytotoxic deletion variant of the Aspergillus fumigatus allergen Aspf1 displaying reduced IgE binding

Aspergillus fumigatus is responsible for many allergic respiratory diseases, the most notable of which - due to its severity - is allergic bronchopulmonary aspergillosis. Aspf1 is a major allergen of this fungus: this 149-amino acid protein belongs to the ribotoxin family, whose best characterized member is alpha-sarcin (EC 3.1.27.10). The proteins of this group are cytotoxic ribonucleases that degrade a unique bond in ribosomal RNA impairing protein biosynthesis. Aspf1 and its deletion mutant Aspf1Delta(7-22) have been produced as recombinant proteins; the deleted region corresponds to an exposed beta-hairpin. The conformation of these two proteins has been studied by CD and fluorescence spectroscopy. Their enzymatic activity and cytotoxicity against human rhabdomyosarcoma cells was also measured and their allergenic properties have been studied by using 58 individual sera of patients sensitized to Aspergillus. Aspf1Delta(7-22) lacks cytotoxicity and shows a remarkably reduced IgE reactivity. From these studies it can be concluded that the deleted beta-hairpin is involved in ribosome recognition and is a significant allergenic region.     

Structurally conserved water molecules in ribonuclease T1

In the high resolution (1.7-1.9 A) crystal structures of ribonuclease T1 (RNase T1) in complex with guanosine, guanosine 2'-phosphate, guanylyl 2',5'-guanosine, and vanadate, there are 30 water sites in nearly identical (+/- 1 A) positions that are considered conserved. One water is tightly bound to Asp76(O delta), Thr93(O gamma), Cys6(O), and Asn9(N); another bridges two loops by hydrogen-bonding to Tyr68(O eta) and to Ser35(N), Asn36(N); a loop structure is stabilized by two waters coordinated to Gly31(O) and His27(N delta), and by water bound to cis-Pro39(O). Most notable is a hydrogen-bonded chain of 10 water molecules. Waters 1-5 of this chain are inaccessible to solvent, are anchored at Trp59(N), and stitch together the loop formed by segments 60-68; waters 5-8 coordinate to Ca2+, and waters 9 and 10 hydrogen-bond to N-terminal side chains of the alpha-helix. The water chain and two conserved water molecules are bound to amino acids adjacent to the active site residues His40, Glu58, Arg77, and His92; they are probably involved in maintaining their spatial orientation required for catalysis. Water sites must be considered in genetic engineering; the mutation Trp59Tyr, which probably influences the 10-water chain, doubles the catalytic activity of RNase T1.     

Ribotoxin recognition of ribosomal RNA and a proposal for the mechanism of translocation.

The ribotoxins alpha-sarcin and ricin catalyse covalent modifications in adjacent nucleotides in 28S rRNA, yet the elements of nucleic acid structure that they recognize are not only different but incompatible. This suggests that this ribosomal domain (which in two dimensions is a seven-base-pair helical stem and a 17-member single-stranded loop) has alternate conformers. Since the domain is involved in binding of aminoacyl-tRNA and GTP hydrolysis, we propose that the switch between the two configurations, perhaps initiated by the binding of elongation factors, plays a role in translocation.     

Alteration of hydrogen bonding in the vicinity of histidine 48 disrupts millisecond motions in RNase A

The motion of amino acid residues on the millisecond (ms) time scale is involved in the tight regulation of catalytic function in numerous enzyme systems. Using a combination of mutational, enzymological, and relaxation-compensated (15)N Carr-Purcell-Meiboom-Gill (CPMG) methods, we have previously established the conformational significance of the distant His48 residue and the neighboring loop 1 in RNase A function. These studies suggested that RNase A relies on an intricate network of hydrogen bonding interactions involved in propagating functionally relevant, long-range ms motions to the catalytic site of the enzyme. To further investigate the dynamic importance of this H-bonding network, this study focuses on the individual replacement of Thr17 and Thr82 with alanine, effectively altering the key H-bonding interactions that connect loop 1 and His48 to the rest of the protein. (15)N CPMG dispersion studies, nuclear magnetic resonance (NMR) chemical shift analysis, and NMR line shape analysis of point mutants T17A and T82A demonstrate that the evolutionarily conserved single H-bond linking His48 to Thr82 is essential for propagating ms motions from His48 to the active site of RNase A on the time scale of catalytic turnover, whereas the T17A mutation increases the off rate and conformational exchange motions in loop 1. Accumulating evidence from our mutational studies indicates that residues experiencing conformational exchange in RNase A can be grouped into two separate clusters displaying distinct dynamical features, which appear to be independently affected by mutation. Overall, this study illuminates how tightly controlled and finely tuned ms motions are in RNase A, suggesting that designed modulation of protein motions may be possible.     

Ribotoxin genes in isolates of Aspergillus section Clavati

Ribotoxins are ribosome inactivator proteins with high specificity against the sarcin/ricin domain of the 28S ribosomal RNA. We examined the presence of ribotoxin genes in isolates of species recently assigned to Aspergillus section Clavati using specific primer pairs. All species assigned to this section have been found to carry ribotoxin genes. Phylogenetic analysis of the sequences of the amplified gene fragments allowed us to classify the genes to different groups including the alpha-sarcin, gigantin, c-sarcin and mitogillin/restrictocin families. Two species, A. longivesica and N. acanthosporus produced ribotoxins which were only distantly related to gigantins and c-sarcins, respectively. Comparison of the protein sequences of the genes to known ribotoxin sequences revealed that all of them carry the presumed catalytic residues of ribotoxins, the cystein residues, and also the two Trp residues of alpha-sarcin conserved in all ribotoxins known so far. These data indicate that these genes probably encode active ribotoxins. Further studies are in progress to examine the secretion and activities of these new ribotoxins.     

Structural properties of the histidine-containing loop in HIV-1 RNase H

The isolated HIV-1 RNase H domain is inactive. This inactivity has been linked to the lack of structure in the C-terminus of the isolated domain. Thermodynamic stability experiments on the RNase H domain as well as a deletion mutant lacking the C-terminal helix have implied that this region is structured. His539 residing in a loop preceding the C-terminal helix was studied by NMR to determine the stability and conformational properties of this region. The stability of the structural environment of His539 matches that of the entire RNase H domain. Furthermore, His539 is locked into a defined tautometric state in the folded protein and its pK(a) is shifted compared to a freely accessible His, suggesting that this region is structured. The data support the view that the overall dynamics rather than the lack of structure in a small portion of the protein render activity of the isolated HIV-1 RNase H.     

The occluding loop in cathepsin B defines the pH dependence of inhibition by its propeptide

Papain-like proenzymes are prone to autoprocess under acidic pH conditions. Similarly, peptides derived from the proregion of cathepsin B are potent pH-dependent inhibitors of that enzyme; i.e., at pH 6.0 the inhibition of human cathepsin B by its propeptide is defined by slow binding kinetics with a Ki of 3.7 nM and at pH 4.0 by classical kinetics with a Ki of 82 nM. This pH dependency is essentially eliminated either by the removal of a portion of the enzyme's occluding loop through deletion mutagenesis or by the mutation of either residue Asp22 or His110 to alanine; e.g., the mutant enzyme His110Ala is inhibited by its propeptide with Ki's of 2.0 +/- 0.3 nM at pH 4.0 and 1.1 +/- 0.2 nM at pH 6.0. For the His110Ala mutant the inhibition also displays slow binding kinetics at both pH 4.0 and pH 6.0. As shown by the crystal structure of mature cathepsin B [Musil, D., et al. (1991) EMBO J. 10, 2321-2330] Asp22 and His110 form a salt bridge in the mature enzyme, and it has been shown that this bridge stabilizes the occluding loop in its closed position [N?gler, D. K., et al. (1997) Biochemistry 36, 12608-12615]. Thus the pH dependency of propeptide binding can be explained on the basis of a competitive binding between the occluding loop and the propeptide. At low pH, when the Asp22-His110 pair forms a salt bridge stabilizing the occluding loop in its closed conformation, the loop more effectively competes with the propeptide than at higher pH where deprotonation of His110 and the concomitant destruction of the Asp22-His110 salt bridge results in a destabilization of the closed form of the loop. The rate of autocatalytic processing of procathepsin B to cathepsin B correlates with the affinity of the enzyme for its propeptide rather than with its catalytic activity, thus suggesting a possible influence of occluding loop stability on the rate of processing.     

Microinjected oligonucleotides complementary to the alpha-sarcin loop of 28 S RNA abolish protein synthesis in Xenopus oocytes

The integrity of the alpha-sarcin loop in 28 S ribosomal RNA is critical during protein synthesis. The toxins alpha-sarcin, ricin, Shiga toxin, and Shiga-like toxin inhibit protein synthesis in oocytes by attacking specific nucleotides within this loop (Ackerman, E.J., Saxena, S. K., and Ulbrich, N. (1988) J. Biol. Chem. 263, 17076-17083; Saxena, S.K., O'Brien, A.D., and Ackerman, E.J. (1989) J. Biol. Chem. 264, 596-601). We injected Xenopus oocytes with deoxyoligonucleotides complementary to the 17-nucleotide alpha-sarcin loop of Xenopus 28 S rRNA. Only injected oligonucleotides fully covering the alpha-sarcin loop or slightly beyond inhibited oocyte protein synthesis. Shorter alpha-sarcin domain deoxyoligonucleotides complementary to the alpha-sarcin and ricin sites but not spanning the entire loop were less effective inhibitors of protein synthesis. The alpha-sarcin domain oligonucleotides covering the entire loop were more effective inhibitors of protein synthesis than injected cycloheximide at equivalent concentrations. Control oligonucleotides complementary to nine other regions of Xenopus 28 S rRNA as well as universal M13 DNA sequencing primers had no effect on oocyte protein synthesis. Oligonucleotides complementary to the highly conserved alpha-sarcin domain therefore represent an alternative to catalytic toxins for causing cell death and may prove effective in immunotherapy.     

The influence of ATP on the binding of aromatic amino acids to the ligand response domain of the tyrosine repressor of Haemophilus influenzae

Mitogillin and related fungal ribotoxins are small basic ribonucleolytic proteins that inhibit protein synthesis by specifically hydrolyzing a single phosphodiester bond in the universally conserved alpha-sarcin/ricin loop (SRL) of large subunit ribosomal RNAs. It was previously shown that mitogillin is a natural derivative of a T1/U2-like ribonuclease with inserted domains that are involved in target selection and specificity. Site-directed mutagenesis was used to substitute single amino acids in the previously identified functional domains Ala1-Tyr24 (B1-L1-B2 domain) and Lys106-Lys113 (L4 region). Examination of the activities of the mutants in the digestion of polyinosinic acid (a ribonuclease substrate) and specific cleavage of the SRL shows that Asn7Ala and Lys111Gln substitutions lead to altered ribonuclease activity and diminished substrate specificity consistent with the proposed functions of these domains.