Roles of Asp179 and Glu270 in ADP-Ribosylation of Actin by Clostridium perfringens Iota Toxin

Abstract

Clostridium perfringens iota toxin is a binary toxin composed of the enzymatically active component Ia and receptor binding component Ib. Ia is an ADP-ribosyltransferase, which modifies Arg177 of actin. The previously determined crystal structure of the actin-Ia complex suggested involvement of Asp179 of actin in the ADP-ribosylation reaction. To gain more insights into the structural requirements of actin to serve as a substrate for toxin-catalyzed ADP-ribosylation, we engineered Saccharomyces cerevisiae strains, in which wild type actin was replaced by actin variants with substitutions in residues located on the Ia-actin interface. Expression of the actin mutant Arg177Lys resulted in complete resistance towards Ia. Actin mutation of Asp179 did not change Ia-induced ADP-ribosylation and growth inhibition of S. cerevisiae. By contrast, substitution of Glu270 of actin inhibited the toxic action of Ia and the ADP-ribosylation of actin. In vitro transcribed/translated human β-actin confirmed the crucial role of Glu270 in ADP-ribosylation of actin by Ia.

Fig 1. Structural representation of actin-Ia interaction.

Upper panel, general view of Ia interaction with actin (pdb code 3BUZ). Lower panel, detailed representation of the region around R177 of actin. Amino acid residues R177, D179, E270, E72 of actin, K351R352K353 motif of Ia and the nonhydrolyzable NAD analog TAD (β-thiazole-4-carboxamide adenine dinucleotide) are shown as sticks. Images were prepared using PyMOL (www.pymol.org).

Fig 2. Analyses of agar growth phenotypes of S. cerevisiae containing different actin variants.

Five-fold serial dilutions of yeast cultures were spotted onto YPD agar. Plates were incubated for 3–4 days at 30°C. Actin variants produced by the corresponding S. cerevisiae strains are shown on the left. Control yeast strain representing wild type S. cerevisiae is indicated as “WT strain”.

Fig 3. Susceptibility of S. cerevisiae containing different actin variants towards iota toxin component Ia.

Yeast strains containing wild type actin or actin variants with substitutions R177K, D179E, D179A, and D179K (panel A) or E270D and E270Q (panel B) were transformed with the vector alone or the Ia-expressing plasmid, and were analyzed by the drop-test under ia-repressing (glucose) or -inducing (galactose) conditions. Plates were incubated for 3–4 days at 30°C.

Fig 4. Analysis of Ia production by engineered S. cerevisiae.

(A) Analysis of the synthesis of Ia in S. cerevisiae strains, producing actin-R177K, E270D and E270Q. Yeast strains, producing actin-R177K, E270D or E270Q and transformed with the Ia-containing plasmid (Ia) or the vector alone (Vector), were cultivated in SGal for 20 h at 30°C. Cells were broken by glass beads treatment and analyzed by ³²P-ADP-ribosylation in the presence of additionally added purified wild type yeast actin (1 μg). Labeled bands represent modified yeast actin and confirm intracellular production of functionally active Ia by the S. cerevisiae strains. (B) Production of Ia by the wild type S. cerevisiae strain. Wild type yeast strains harboring the Ia-containing plasmid (Ia) or the control vector (vector) were cultivated in glucose-containing liquid medium until OD₅₉₅ = 0.5. Afterwards, glucose was replaced by galactose and cultivation continued for 9 h at 30°C. Cells were lysed and the resulting extract preparations were ADP-ribosylated in the presence of Ia (+ Ia), TccC3 toxin of P. luminescens [42] (+ TccC3), purified muscle actin (+α-actin) or tested in Western blotting with the anti-actin serum to show equal actin concentrations in the samples. (C, D) Mass spectrometry of actin variants. MALDI-TOF MS of wild type (C) and actin-R177K (D) protein variants isolated from S. cerevisiae. Spectra demonstrate disappearance of R177- and appearance of K177-containing peptide in mass analysis (substituted amino acid residue within identified peptides is shown in red).

Fig 5. In vitro ADP-ribosylation of actin variants produced by S. cerevisiae.

(A) Yeast extracts were prepared from strains producing wild type actin or actin variants with substitutions R177K, D179A, E270D or E270Q and were tested in ³²P-ADP-ribosylation with Ia or C. difficile transferase CDT-A. (B) Time course of ³²P-ADP-ribosylation performed in the presence of Ia with wild-type actin (◆) or actin variants D179E (△), D179A (□), D179K (○) or R177K (●). Means of three measurements with standard deviation are shown. (C) Time course of ³²P-ADP-ribosylation performed in the presence of Ia with wild type actin (◆) or actin variants E270Q (△), E270D (□). Means of three measurements with standard deviation are shown.

Fig 6. In vitro ADP-ribosylation of human β-actin variants.

Actin variants were produced in in vitro transcription/translation reaction, using as a matrix plasmids coding for human β-actin gene with the corresponding amino acid substitutions (wild type (WT), R177K, D179A, E270D and E270Q). Afterwards, 1 μl of the in vitro transcription/translation mix was ADP-ribosylated with Ia (150 ng/10 μl in Panel A; 15 and 50 ng/10 μl in Panel B; and 15, 50 or 150 ng/10 μl in Panel C) or left untreated, without toxin (w/o). Reaction mixes were subjected to non-denaturing polyacrylamide gel electrophoresis and autoradiography (shown) to detect ³⁵S-methionine-labelled actin variants. Arrows on the left indicate position of shifted ADP-ribosylated actin.