Endogenous ADP-ribosylation for eukaryotic elongation factor 2: evidence of two different sites and reactions

2006 ◽  
Vol 24 (4) ◽  
pp. 369-380 ◽  
Author(s):  
Muhammet Bektaş ◽  
Rüstem Nurten ◽  
Kıvanç Ergen ◽  
Engin Bermek
Keyword(s):  
Elongation Factor ◽  
Adp Ribosylation ◽  
Elongation Factor 2 ◽  
Eukaryotic Elongation Factor 2 ◽  
Eukaryotic Elongation Factor

The role of the diphthamide-containing loop within eukaryotic elongation factor 2 in ADP-ribosylation by Pseudomonas aeruginosa exotoxin A

2008 ◽  
Vol 413 (1) ◽  
pp. 163-174 ◽  
Author(s):  
Yong Zhang ◽  
Suya Liu ◽  
Gilles Lajoie ◽  
A. Rod Merrill
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Elongation Factor ◽  
Exotoxin A ◽  
Mutant Proteins ◽  
Adp Ribosylation ◽  
Elongation Factor 2 ◽  
Eukaryotic Elongation Factor 2 ◽  
Eukaryotic Elongation Factor ◽  
Pseudomonas Aeruginosa Exotoxin

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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