1. Centre for Structural Biology, Department of Molecular Biology, University of Aarhus, Gustav Wieds Vej 10C, DK-8000, Denmark
2. Department of Molecular and Cellular Biology, and
3. Department of Chemistry, University of Guelph, Guelph, Ontario N1G 2W1, Canada
4. Laboratory of Medicinal Chemistry, Center for Cancer Research, National Cancer Institute at Frederick, NIH, Frederick, Maryland 21702, USA
5. *These authors contributed equally to this work

Correspondence to: A. Rod Merrill^2,5Gregers R. Andersen¹ Correspondence and requests for materials should be addressed to G.R.A. (Email: gra@mb.au.dk) or A.R.M. (Email: rmerrill@uoguelph.ca). Coordinates and structure factors are deposited at the RCSB data bank as entries 1ZM2, 1ZM3, 1ZM4 and 1ZM9.

Abstract

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, TAD. 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 TAD 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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