Systematic Deletion Analysis of Ricin A-Chain Function

Abstract

The A-chain of ricin is a cytotoxic RNA N-glycosidase that inactivates ribosomes by depurination of the adenosine at position 4324 in 28 S rRNA. Of the 267 amino acids in the protein, 222 could be deleted, in one or another of 74 mutants, without the loss of the capacity to catalyze hydrolysis of a single specific nucleotide in rRNA (Morris, K. N., and Wool, I. G. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 4869 -4873). The 45 amino acids that could not be omitted when the deletions were in sets of 20, 5, or 2 residues have now been deleted one at a time; 9 of these deletion mutants retained activity. A RNP-like structural motif in ricin A-chain that may mediate binding to ribosomal RNA has been identified.Ricin is a cytotoxic RNA N-glycosidase that is synthesized in the castor bean Ricinus communis. Proricin is a polypeptide of approximately 65 kDa which is processed by removal of 12 amino acids to form an A-chain of 267, and a B-chain of 262, residues linked by a disulfide bond (1). The toxic ricin A-chain (RA) 1 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 28 S rRNA (2, 3). RA is extraordinarily toxic: a single molecule will inactivate 1500 ribosomes min Ϫ1, indeed, a single molecule resident in a cell is sufficient to kill it (1); finally, this one covalent modification accounts entirely for the cytotoxicity.A value of an analysis of the mechanism of action of antibiotics and of ribotoxins is that it directs attention to components of the ribosome where efforts to comprehend functional correlates of structure are likely to be rewarded. That this one RA-catalyzed depurination inactivates the ribosome implies that this region of 28 S rRNA is crucial for the function of the particle and there is evidence that the ricin domain is involved in elongation factor-1 dependent binding of aminoacyl-tRNA to the ribosomal A-site and elongation factor-2 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 (6 -15) and an atomic structure of ricin has been obtained from x-ray diffraction of crystals (16 -18). In the three-dimensional structure there is a prominent cleft that was proposed to be the active site of RA before the mechanism of action of the toxin had been defined, indeed, before the substrate had been identified (16). Strong support for the suggestion has come from the observation that most of the amino acids that are invariant in related plant and bacterial toxins are clustered at the bottom of the putative active site cleft (18); from site-directed mutagenesis (19 -24); and from an analysis by systematic deletion of amino acids (25).Three series of amino acid deletion...

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

“…In previous studies, various random mutagenesis methods have been used to isolate nontoxic RTA mutants from yeast (1,10). Systematic deletion analysis has been used to identify amino acids critical for the activity of RTA in vitro (18,31). Our study is the first report which determines the precise effects of each mutation on cytotoxicity, ribosome depurination, and translation inhibition and provides evidence that cytotoxicity of RTA is not entirely due to ribosome depurination.…”

“…The E177K mutation was isolated several times in different studies (1,10). Mutations in Arg180 (R180G) (1) in helix E and Ile184 (⌬I184) (31) at the beginning of helix F disrupted the enzymatic activity of RTA in vitro, emphasizing the critical nature of this region. In our study, deletion of Ile184 led to a loss of cytotoxicity and ribosome depurination in vivo (Table 1).…”

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

“…The mechanism was also suggested by the results of site-directed mutagenesis [16Ϫ21] and an analysis by systematic deletions of amino acids [22,23]. Wool and co-workers had studied the role of amino acids in RA protein on the activity using systematic deletions [22,23]. In the amino acid deletion experiments, four series of consecutive omissions of 20, 5, 2 or 1 amino acid(s) that scanned RA were constructed.…”

“…Appearance of RA fragment (460 nucleotides) in gel electrophoresis by release from RNA extracted from reticulocyte ribosomes was an indicator of the activity. 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].…”

“…Wool and co-workers had studied the role of amino acids in RA protein on the activity using systematic deletions [22,23]. In the amino acid deletion experiments, four series of consecutive omissions of 20, 5, 2 or 1 amino acid(s) that scanned RA were constructed.…”