Structure, mechanism, and specificity of a eukaryal tRNA restriction enzyme involved in self-nonself discrimination

Abstract

tRNA restriction by anticodon nucleases underlies cellular stress responses and self-nonself discrimination in a wide range of taxa. Anticodon breakage inhibits protein synthesis, which, in turn, results in growth arrest or cell death. The eukaryal ribotoxin PaT secreted by Pichia acaciae inhibits growth of Saccharomyces cerevisiae via cleavage of tRNA(Gln(UUG)). We find that recombinant PaT incises a synthetic tRNA(Gln(UUG)) stem-loop RNA by transesterification at a single site 3' of the wobble uridine, yielding 2',3'-cyclic phosphate and 5'-OH ends. Incision is suppressed by replacement of the wobble nucleobase with adenine or guanine. The crystal structure of PaT reveals a distinctive fold and active site, essential components of which are demonstrated by mutagenesis. Pichia acaciae evades self-toxicity via a distinctive intracellular immunity protein, ImmPaT, which binds PaT and blocks nuclease activity. Our results highlight the evolutionary diversity of tRNA restriction and immunity systems.

Fig. 1. PaT is toxic in vivo and incises a synthetic tRNA^Gln stem-loop in vitro at a unique site 3′ of the wobble uridine

(A) Secreted tRNA ribotoxins defend fungi against non-self species. Pichia acaciae harbors a killer plasmid episome that encodes a secreted heterodimeric toxin. The chitin-binding Orf1 subunit interacts with the surface of S. cerevisiae target cells and mediates delivery of the toxic PaT (Orf2) subunit into the cytoplasm, where it arrests the growth of the target cell. (B) PaT is a tRNA anticodon nuclease that incises tRNAGln^(UUG). (C) Ribotoxicity assay. Schematic depiction of yeast cells harboring the pGAL-PaT plasmid (2μ LEU2) in the presence of glucose or galactose as the carbon source. In this experment, the N-terminal peptide sequence of PaT was varied as indicated on the left. The reference N-terminal sequence of the unprocessed PaOrf2 protein is shown at bottom, with the presumed signal peptide in lower case and colored blue. By convention, the asparagine is designated amino acid 2 of the intracellular PaT (Meineke et al., 2012). Aliquots of serial dilutions of the indicated S. cerevisiae pGAL-PaT strains were spotted on SD-Leu^– agar containing 2% glucose or 2% galactose. (D) Purified recombinant PaT. An aliquot (6 μg) of purified gel-filtered PaT–His₆ was analyzed by SDS-PAGE. The Coomassie blue-stained gel is shown. The positions and sizes (kDa) of marker polypeptides are indicated on the right. (E) Anticodon nuclease activity. RNase reaction mixtures (10 μl) containing 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM DTT, 0.1 μM 5′ ³²P-labeled 27-mer stem-loop RNA (shown at right with the UUG anticodon colored magenta and the 5′-labeled nucleotide in red) and either no protein (lane –) or 0.063, 0.125, 0.25 0.5 1, or 2 μM purified PaT–His₆ (increasing from left to right) were incubated at 30°C for 60 min. The products were analyzed by urea-PAGE in parallel with size marker ladders generated by: (i) partial alkaline hydrolysis of the same 5′ ³²P-labeled 27-mer RNA substrate (lane OH); (ii) partial alkaline hydrolysis of an otherwise identical 27-mer in which the wobble nucleoside was U_2′F and thereby alkali-resistant (OH*); and (iii) treatment of the ³²P-labeled 27-mer RNA with RNase T1, which cleaves 3′ of the lone unpaired guanosine in the anitcodon loop to yield a 5′-labeled 15-mer with a 3′-phosphate end. An autoradiograph of the gel is shown; the identities of the 3′ terminal nucleotides are indicated next to the ladder. See also Figures S1 and S2.

Fig. 2. Effect of wobble base substitutions and ribose modification

(A) Reaction mixtures (10 μl) containing 0.1 μM 5′ ³²P-labeled 27-mer stem-loop RNA with the wobble nucleoside as specified (U, U_2′F, A, C or G) and either 2 μM PaT (lanes +) or no enzyme (lanes –) were incubated at 30°C for 60 min. The products were analyzed by urea-PAGE in parallel with partial alkaline hydrolysates of each of the ³²P-labeled 27-mer RNAs (lanes OH). The species in each alkaline ladder corresponding to the 5′-fragment cleaved 3′ of the wobble nucleoside is denoted by ●. (B) The extents and sites of incision by PaT of the 27-mer RNAs with U, C, A or G wobble nucleosides are plotted in bar graph format, where the anticodon loop sequences are arrayed on the x-axes, with the variable wobble nucleobases highlighted in the gray boxes. Each datum in the graphs is the average of three separate experiments ±SEM. Note that the y-axis scales are different in each graph.

Fig.3. Crystal structure of PaT reveals a unique fold

(A) The tertiary structure of PaT–His₆ is shown as a ribbon trace with magenta β strands, cyan α helices, blue 3₁₀ helices, and beige intervening loops and turns. The N and C termini of the polypeptide are indicated; selected α helices and all β strands are numbered according to their order in the primary structure. (B) The surface model of PaT in the same orientation as panel A and its vacuum electrostatics were generated in Pymol. Two sulfate anions coordinated on the protein surface are shown as stick models. A chloride anion is shown as a green sphere. (C) The aligned primary structures of the Pichia acaciae (Pac) PaT polypeptide and the homologous toxin from Debaryomyces robertsiae (Dro) are shown. Positions of side chain identity/similarity are denoted by ● above the sequence. Gaps in the alignment are indicated by dashes. The secondary structure elements of PaT are shown above the amino acid sequence, with β strands rendered as arrows and helices as cylinders, colored as in panel A. The amino acids in PaT that were subjected to alanine scanning in the present study are highlighted in shaded boxes: in green for the essential residues and gray for the nonessential residues. See also Figure S4.

Fig. 4. Distinctive active site of PaT

(A) Stereo view of the putative active site of PaT–His₆ with selected amino acid side chains and main chain atoms depicted as stick models with beige carbons. A sulfate anion is rendered as a stick model; a chloride anion is depicted as a green sphere. Atomic contacts are indicated by dashed lines. Site side chains found, by alanine scanning, to be essential for toxicity are denoted by red labels. (B) Effects of alanine mutations on in vivo toxicity. Aliquots of serial dilutions of the S. cerevisiae pGAL-PaT strains, expressing wild-type PaT or the indicated alanine mutants, were spotted on SD-Leu^– agar containing 2% glucose or 2% galactose. Cells carrying the empty 2μ vector were tested in parallel as controls. Essential residues were those at which alanine substitution permitted growth on galactose. (C) Effects of selected PaT mutations on anticodon nuclease activity in vitro. Aliquots (5 μg) of the SP-Sepharose preparations of wild type PaT–His₆ and the R172A, K175A, S283A and H287A mutants were analyzed by SDS-PAGE. The Coomassie blue-stained gel is shown in the left panel. The PaT–His₆ proteins (2 μM) were reacted with the 5′ ³²P-labeled 27-mer stem-loop RNA (0.1 μM) and 2 M TMAO for 60 min at 30°C. The extents of anticodon cleavage are plotted in the bar graph in the right panel; each datum is the average of three separate experiments ±SEM. See also Figure S3.

Fig. 5. Pichia acaciae toxin immunity protein (ImmPaT) binds PaT in vitro and effaces its endoribonuclease activity

(A) Recombinant ImmPaT. An aliquot (6 μg) of purified ImmPaT was analyzed by SDS-PAGE. The Coomassie blue-stained gel is shown. The ImmPaT polypeptide is denoted by ◄. The positions and sizes (kDa) of marker polypeptides are indicated on the left. (B) ImmPaT inhibits PaT anticodon nuclease activity. PaT–His₆ and either ImmPaT or BSA in amounts specified were preincubated for 5 min at 30°C, then reacted with 0.1 μM (1 pmol) of 5′ ³²P-labeled 27-mer stem-loop RNA for 60 min at 30°C. The products were analyzed by urea-PAGE and visualized by autoradiography. (C and D) PaT•ImmPaT complex formation. PaT–His₆ alone, ImmPaT alone, and a mixture of PaT–His₆ and ImmPaT (1:1.5 molar ratio; preincubated for 5 min at 30°C) were gel-filtered through a 25-ml Superdex 200 column in 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM DTT. The superimposed chromatographic profiles (monitored by A₂₈₀ as a function of elution volume) are shown as solid lines in panel C. The dotted line in panel C depicts the elution profile of a mixture of ferritin, aldolase, conalbumin, ovalbumin, and carbonic anhydrase, with native molecular weights (kDa) indicated by arrows, that was used to calibrate the column. Panel D shows the polypeptide composition of the serial column fractions from the gel-filtration profile of the mixture of PaT–His₆ and ImmPaT. The fractions corresponding to the PaT•ImmPaT complex are indicated by the bracket. (E) The PaT•ImmPaT complex has no anticodon nuclease activity. 5′ ³²P-labeled 27-mer stem-loop RNA (20 nM) was reacted with either 0.4 μM PaT–His₆ or PaT•ImmPaT complex (from the peak Superdex fraction denoted by ● in panel D), for 60 min at 30°C. The products were analyzed by urea-PAGE and visualized by autoradiography.