Role of arginine 180 and glutamic acid 177 of ricin toxin A chain in enzymatic inactivation of ribosomes.

Frankel, Arthur E.; Welsh, Phil; Richardson, Jane; Robertus, Jon D.

doi:10.1128/mcb.10.12.6257

Cited by 92 publications

(79 citation statements)

References 28 publications

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“…Glu177, Arg180, Tyr80, Tyr123, Trp211 and Asn209) [16,17,20]. To date, the principle mechanism of RA action is thought to be as follows.…”

Section: Discussionmentioning

confidence: 99%

Involvement of the amino acids outside the active‐site cleft in the catalysis of ricin A chain

Kitaoka

1998

European Journal of Biochemistry

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The A chain of ricin (RA) is a cytotoxic RNA N-glycosidase that inactivates ribosomes by depurination of the adenosine residue at position 4324 in 28S rRNA. Of the 267 amino acids in the protein, 231 could be deleted in one or another of 83 mutants, without the loss of the capacity to catalyze hydrolysis of a single specific nucleotide in rRNA [Morris, K. N. & Wool, I. G. (1992) Proc. Natl Acad. Sci. USA 89, 4869Ϫ4873]. Expression of 29 selected deletion mutants of RA in prokaryotic cell-free coupled transcription-translation reactions was carried out and the activities of the mutants were assessed by monitoring depurination of reticulocyte ribosomes. Kinetic analysis of five deletion mutants which retained detectable activity was performed. Deletion of amino acids outside the putative active-site cleft in these mutants significantly affected the catalytic rate rather than the interaction with ribosomes. From these data, the amino acids far from the active-site cleft appeared to be involved in alignment of the key residues for catalysis and interaction with the target tetraloop structure of 28S rRNA.Keywords : ricin; N-glycosidase; cell-free coupled transcription-translation; ribosome; protein synthesis.Ricin is a cytotoxic RNA N-glycosidase that is found in the seeds of Ricinus communis. Proricin is processed by removal of a connecting peptide of 12 amino acids to form an A chain of 267 residues and a B chain of 262 residues linked by a disulfide bond [1]. The toxic ricin A chain (RA) inhibits protein synthesis by inactivating ribosomes ; the inhibition is the result of the hydrolysis of the bond between the base and the ribose of the adenosine at position 4324 (A4324) in 28S rRNA [2,3]. RA is extraordinarily toxic; and a single molecule will inactivate 1500 ribosomes min Ϫ1 ; indeed, a single molecule is sufficient to kill a cell [1] and this one covalent modification accounts entirely for the cytotoxicity.The value of an analysis of the mechanism of action of ribotoxins is that it directs attention to components of the ribosome where efforts to comprehend functional correlates of the structure are likely to be rewarded. That this one RA-catalyzed depurination inactivates the ribosome implies that this region of 28S rRNA is crucial for the function of the particle. There is evidence that the ricin domain is involved in elongation-factor-1-(or Tu)-dependent binding of aminoacyl-tRNA to the ribosomal A site and elongation-factor-2-(or G)-catalyzed GTP hydrolysis and translocation of peptidyl-tRNA to the P site [4].A substantial effort has been made to relate the structure of RA to its mechanism of action spurred in part by the use of the protein in the construction of immunotoxins for cancer therapy [5]. The amino acid sequences of ricin and of several homologous proteins have been determined [6Ϫ12] and an atomic structure of ricin has been obtained from X-ray diffraction of crystals [13Ϫ15]. In the three-dimensional structure, there is a prominent cleft that was proposed to be the active site of RA before the ...

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“…Glu177, Arg180, Tyr80, Tyr123, Trp211 and Asn209) [16,17,20]. To date, the principle mechanism of RA action is thought to be as follows.…”

Section: Discussionmentioning

confidence: 99%

Involvement of the amino acids outside the active‐site cleft in the catalysis of ricin A chain

Kitaoka

1998

European Journal of Biochemistry

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“…However, PAP II had a similar region at positions 159 to 184. This relatively well conserved region is thought to be the active site of enzymatic activity of the RIPS [22,23]. It has been shown for the ricin A-chain that two amino acids of these relatively conserved sequences are involved in the catalytic activity [22,23].…”

Section: Sequence Comparisonmentioning

confidence: 99%

Isolation and characterization of a cDNA clone encoding the pokeweed antiviral protein II from Phytolacca americana and its expression in E. coli

1994

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Three distinct ribosome-inactivating proteins (RIPS) were isolated from pokeweed (Phytolacca americana). We identified and sequenced for the first time a complete cDNA encoding the pokeweed antiviral protein II (PAP II), which is expressed in the late summer leaves of pokewecd. The cDNA of PAP II consists of 1,187 nucleotides and encodes a mature protein of 285 amino acids. Its predicted amino acid sequence is only 33% similar to PAP and PAP-S. The NH2 terminal extrapeptide (25 amino acid residues) was similar but not identical to that of PAP's extrapeptide. The cDNA of PAP II was expressed in E. coli. The growth of the transformants was strongly inhibited after induction of the gene. Furthermore, PAP II, which was produced in E. coli, inhibited protein synthesis in a rabbit reticulocyte translation system. Thus, recombinant PAP II would appear to be as functional as native PAP in inhibiting protein synthesis in both prokaryotes and eukaryotes.

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“…15 The X-ray structure of intact heterodimeric ricin is known, 16 and the enzymatic A chain of ricin, called RTA, has been described at higher resolution. 14,17,18 Sitedirected mutagenesis, kinetic studies, 19,20 and the binding of substrate analogues to the crystallaline RTA 21 have provided crucial information about the mechanism of action. The adenine base binds in a specificity pocket between the side chains of tyrosines 80 and 123 and makes specific hydrogen bonds with the protein.…”

Section: Introductionmentioning

confidence: 99%

Structure-Based Design and Characterization of Novel Platforms for Ricin and Shiga Toxin Inhibition

et al. 2001

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Ribosome inhibiting proteins, RIPs, are a widespread family of toxic enzymes. Ricin is a plant toxin used as a poison and biological warfare agent; shiga toxin is a homologue expressed by pathogenic strains of E. coli. There is interest in creating effective antidote inhibitors to this class of enzymes. RIPs act by binding and hydrolyzing a specific adenine base from rRNA. Previous virtual screens revealed that pterins could bind in the specificity pocket of ricin and inhibit the enzyme. In this paper we explore a range of compounds that could serve as better platforms for inhibitor design. This establishes the importance of key hydrogen bond donors and acceptors for active-site complementarity. 8-Methyl-9-oxoguanine is a soluble compound that has the best inhibitory properties of any platform tested. The X-ray structure of this complex revealed that the inhibitor binds in an unexpected way that provides insight for future design. Several inhibitors of ricin were also shown to be inhibitors of shiga toxin, suggesting this program has the potential to develop effective antidotes to an important form of food poisoning.

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Role of arginine 180 and glutamic acid 177 of ricin toxin A chain in enzymatic inactivation of ribosomes.

Cited by 92 publications

References 28 publications

Involvement of the amino acids outside the active‐site cleft in the catalysis of ricin A chain

Involvement of the amino acids outside the active‐site cleft in the catalysis of ricin A chain

Isolation and characterization of a cDNA clone encoding the pokeweed antiviral protein II from Phytolacca americana and its expression in E. coli

Structure-Based Design and Characterization of Novel Platforms for Ricin and Shiga Toxin Inhibition

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