eEF2 (eukaryotic elongation factor 2) contains a post-translationally modified histidine residue, known as diphthamide, which is the specific ADP-ribosylation target of diphtheria toxin, cholix toxin and Pseudomonas aeruginosa exotoxin A. Site-directed mutagenesis was conducted on residues within the diphthamide-containing loop (Leu693–Gly703) of eEF2 by replacement with alanine. The purified yeast eEF2 mutant proteins were then investigated to determine the role of this loop region in ADP-ribose acceptor activity of elongation factor 2 as catalysed by exotoxin A. A number of single alanine substitutions in the diphthamide-containing loop caused a significant reduction in the eEF2 ADP-ribose acceptor activities, including two strictly conserved residues, His694 and Asp696. Analysis by MS revealed that all of these mutant proteins lacked the 2′-modification on the His699 residue and that eEF2 is acetylated at Lys509. Furthermore, it was revealed that the imidazole ring of Diph699 (diphthamide at position 699) still functions as an ADP-ribose acceptor (albeit poorly), even without the diphthamide modification on the His699. Therefore, this diphthamide-containing loop plays an important role in the ADP-ribosylation of eEF2 catalysed by toxin and also for modification of His699 by the endogenous diphthamide modification machinery.
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Research Article|
June 12 2008
The role of the diphthamide-containing loop within eukaryotic elongation factor 2 in ADP-ribosylation by Pseudomonas aeruginosa exotoxin A
Yong Zhang;
†Department of Molecular and Cellular Biology, Biophysics Interdepartmental Group, University of Guelph, Guelph, ON, Canada N1G 2W1
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Suya Liu;
Suya Liu
1
*Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada N6A 5C1
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Gilles Lajoie;
Gilles Lajoie
*Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada N6A 5C1
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A. Rod Merrill
A. Rod Merrill
3
†Department of Molecular and Cellular Biology, Biophysics Interdepartmental Group, University of Guelph, Guelph, ON, Canada N1G 2W1
3To whom correspondence should be addressed (email rmerrill@uoguelph.ca).
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Publisher: Portland Press Ltd
Received:
August 09 2007
Revision Received:
March 20 2008
Accepted:
March 28 2008
Accepted Manuscript online:
March 28 2008
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2008 Biochemical Society
2008
Biochem J (2008) 413 (1): 163–174.
Article history
Received:
August 09 2007
Revision Received:
March 20 2008
Accepted:
March 28 2008
Accepted Manuscript online:
March 28 2008
Citation
Yong Zhang, Suya Liu, Gilles Lajoie, A. Rod Merrill; The role of the diphthamide-containing loop within eukaryotic elongation factor 2 in ADP-ribosylation by Pseudomonas aeruginosa exotoxin A. Biochem J 1 July 2008; 413 (1): 163–174. doi: https://doi.org/10.1042/BJ20071083
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