Analysis of the contribution of an amphiphilic alpha-helix to the structure and to the function of ricin A chain.

Morris, Kevin N.; Wool, Ira G.

doi:10.1073/pnas.91.16.7530

Cited by 13 publications

(9 citation statements)

References 20 publications

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“…7). Each of the amino acids in helix D could be singly deleted, provided that the deletion does not disrupt the amphipathicity of the helix (29). The A147P mutation reduced the depurination activity of RTA and led to the loss of its cytotoxicity ( Table 1).…”

Section: Discussionmentioning

confidence: 99%

Ribosome Depurination Is Not Sufficient for Ricin-Mediated Cell Death in Saccharomyces cerevisiae

Baricevic

Saidasan

et al. 2007

Infect Immun

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The plant toxin ricin is one of the most potent and lethal substances known. Ricin inhibits protein synthesis by removing a specific adenine from the highly conserved ␣-sarcin/ricin loop in the large rRNA. Very little is known about how ricin interacts with ribosomes and the molecular mechanism by which it kills cells. To gain insight to the mechanism of ricin-induced cell death, we set up yeast (Saccharomyces cerevisiae) as a simple and genetically tractable system to isolate mutants defective in cytotoxicity. Ribosomes were depurinated in yeast cells expressing the precursor form of the A chain of ricin (pre-RTA), and these cells displayed apoptotic markers such as nuclear fragmentation, chromatin condensation, and accumulation of reactive oxygen species. We conducted a large-scale mutagenesis of pre-RTA and isolated a panel of nontoxic RTA mutants based on their inability to kill yeast cells. Several nontoxic RTA mutants depurinated ribosomes and inhibited translation to the same extent as wild-type RTA in vivo. The mutant proteins isolated from yeast depurinated ribosomes in vitro, indicating that they were catalytically active. However, cells expressing these mutants did not display hallmarks of apoptosis. These results provide the first evidence that the ability to depurinate ribosomes and inhibit translation does not always correlate with ricin-mediated cell death, indicating that ribosome depurination and translation inhibition do not account entirely for the cytotoxicity of ricin.

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Section: Discussionmentioning

confidence: 99%

Ribosome Depurination Is Not Sufficient for Ricin-Mediated Cell Death in Saccharomyces cerevisiae

Baricevic

Saidasan

et al. 2007

Infect Immun

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“…Of 183 mutants, 83 retained the capacity to catalyze depurination of A4324 and, in the latter, 231 of the 267 residues in RA were omitted [22,23]. Four additional amino acids could be omitted if care was taken to construct the deletions so that the amphiphilic character of helix D was preserved [24]. Since the RA protein was not isolated, it was not possible to determine the enzymatic activity exactly.…”

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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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“…The hydrophobic δ-helix in ricin A, also termed helix E, is shielded from solvent by the amphipathic helix D that turns its hydrophobic face toward helix E and its hydrophilic face toward the solvent. 13 In this case, it is apparently a necessary feature in the active-site architecture (in concert with interactions with the ribosome) that charged residues in the δ-helix are placed on a hydrophobic scaffold.…”

Section: δ-Helix Hydrophobicitymentioning

confidence: 98%

Converting a Marginally Hydrophobic Soluble Protein into a Membrane Protein

Nørholm

Cunningham

Deber

et al. 2011

Journal of Molecular Biology

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Analysis of the contribution of an amphiphilic alpha-helix to the structure and to the function of ricin A chain.

Cited by 13 publications

References 20 publications

Ribosome Depurination Is Not Sufficient for Ricin-Mediated Cell Death in Saccharomyces cerevisiae

Ribosome Depurination Is Not Sufficient for Ricin-Mediated Cell Death in Saccharomyces cerevisiae

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

Converting a Marginally Hydrophobic Soluble Protein into a Membrane Protein

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