Insights into diphthamide, key diphtheria toxin effector

Abstract

Diphtheria toxin (DT) inhibits eukaryotic translation elongation factor 2 (eEF2) by ADP-ribosylation in a fashion that requires diphthamide, a modified histidine residue on eEF2. In budding yeast, diphthamide formation involves seven genes, DPH1-DPH7. In an effort to further study diphthamide synthesis and interrelation among the Dph proteins, we found, by expression in E. coli and co-immune precipitation in yeast, that Dph1 and Dph2 interact and that they form a complex with Dph3. Protein-protein interaction mapping shows that Dph1-Dph3 complex formation can be dissected by progressive DPH1 gene truncations. This identifies N- and C-terminal domains on Dph1 that are crucial for diphthamide synthesis, DT action and cytotoxicity of sordarin, another microbial eEF2 inhibitor. Intriguingly, dph1 truncation mutants are sensitive to overexpression of DPH5, the gene necessary to synthesize diphthine from the first diphthamide pathway intermediate produced by Dph1-Dph3. This is in stark contrast to dph6 mutants, which also lack the ability to form diphthamide but are resistant to growth inhibition by excess Dph5 levels. As judged from site-specific mutagenesis, the amidation reaction itself relies on a conserved ATP binding domain in Dph6 that, when altered, blocks diphthamide formation and confers resistance to eEF2 inhibition by sordarin.

Figure 1

Diphthamide synthesis on yeast translation elongation factor 2 (eEF2) and ADP-ribosylation by diphtheria toxin (DT). For details, see text.

Figure 2

(His)₆-tagged Dph1 and Dph2 expressed from E. coli interact with each other in vitro. (A) Anti-(His)₆ Western blot following denaturing conditions (12% SDS-PAGE). (B) Western blot under native conditions (10% native PAGE, 0.5 × TBE).

Figure 3

Use of DT and sordarin as diagnostic tools to map Dph1 regions crucial for Dph2 and Dph3 interaction. (A) Diagram illustrating the N- and C-terminal Dph1 truncation sets (Figure S1) used to study Dph1 function and interaction profiles. (B) DT and sordarin sensitivity assays. Serial cell dilutions of wild-type (wt), DPH1 deletant (dph1Δ) and the strains indicated in panel A were grown in the absence (control) or presence of DT or sordarin. ‘S’ and ‘R’ denote sensitive and resistant traits, respectively. (C, D) Anti-c-Myc co-immune precipitation (IP) assays to study Dph1-Dph2 and Dph1-Dph3 protein-protein interactions. The presence of c-Myc-tagged Dph2 (panel C), Dph3 (panel D), the HA-tagged full-length Dph1 (N, C) and the N- and C-terminal truncation variants of Dph1 in the IPs were monitored by anti-c-Myc and anti-HA Western blots. In addition, the content of full-length and truncated forms of HA-tagged Dph1 was checked by immune blots in the inputs (pre-IP). The positions of Dph2, Dph3 as well as full-length and truncation forms of Dph1 are indicated by arrows.

Figure 4

Overexpression of DPH5 is growth inhibitory to dph1 truncation and deletion mutants. Strains with the indicated genetic backgrounds (see Figure 3) and maintaining plasmid pGAL-DPH5 for galactose inducible overexpression of Dph5 were serially diluted and spotted onto glucose (2% glc) and galactose (2% gal) media to assay their response to DPH5 overexpression. Unaltered tolerance (T) and sensitive (S) responses are indicated.

Figure 5

DPH6 mutagenesis identifies domains in Dph6 that are essential for its function in sordarin sensitivity and dipthamide synthesis. (A) Diagram showing the DPH6 wild-type and mutant constructs tested in (B), indicating the Alpha_ANH_like_IV (ANH_IV: red) and YjgF-YER057c-UK114 (UK114: blue) domains and the position of point mutations. (B) Ten-fold serial cell dilutions of a dph6Δ deletion strain carrying the constructs shown in (A) or the corresponding empty vector pSU6 were grown onto plates with or without sordarin. ‘S’ and ‘R’ denote sensitive and resistant traits, respectively.