The bacteria causing diphtheria, whooping cough, cholera and other diseases secrete mono-ADP-ribosylating toxins that modify intracellular proteins. Here, we describe four structures of a catalytically active complex between a fragment of Pseudomonas aeruginosa exotoxin A (ETA) and its protein substrate, translation elongation factor 2 (eEF2). The target residue in eEF2, diphthamide (a modified histidine), spans across a cleft and faces the two phosphates and a ribose of the non-hydrolysable NAD+ analogue, βTAD. This suggests that the diphthamide is involved in triggering NAD+ cleavage and interacting with the proposed oxacarbenium intermediate during the nucleophilic substitution reaction, explaining the requirement of diphthamide for ADP ribosylation. Diphtheria toxin may recognize eEF2 in a manner similar to ETA. Notably, the toxin-bound βTAD phosphates mimic the phosphate backbone of two nucleotides in a conformational switch of 18S rRNA, thereby achieving universal recognition of eEF2 by ETA.
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G.R.A. was supported by the Benzon Foundation, HFSP, EU FP5 and SNF. A.R.M. was supported by CIHR and CCFF. We are grateful to R. Søe for purification of eEF2, and to J. Nyborg and D. Brodersen for reading of the manuscript. Author Contributions R.J., A.R.M., V.E.M. and G.R.A. performed the experimental work, whereas S.P.Y., A.L.S. and T.B. analysed data.
Coordinates and structure factors are deposited at the RCSB data bank as entries 1ZM2, 1ZM3, 1ZM4 and 1ZM9. Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.
Legends to accompany Supplementary Figures S1-S3.
Electron densities for TAD, the ADP-ribosylation, and PJ34.
Cartoon representations of the four toxin targets G s, RhoA, Actin and G i showing the positions of the ADP-ribosylated residues and the proposed Asp696 (D) and diphthamide (DA) analogues.
A putative function of the diphthamide in translocation.
Statistics for data collection and refinement of the two structures.
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Jørgensen, R., Merrill, A., Yates, S. et al. Exotoxin A–eEF2 complex structure indicates ADP ribosylation by ribosome mimicry. Nature 436, 979–984 (2005). https://doi.org/10.1038/nature03871
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