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.

scholarly journals A re-evaluation of the role of histidine-426 within Pseudomonas aeruginosa exotoxin A

2002 ◽  
Vol 367 (3) ◽  
pp. 601-608 ◽  
Author(s):  
Tania M. ROBERTS ◽  
A. Rod MERRILL
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Residue Analysis ◽  
Elongation Factor ◽  
Protein Translation ◽  
Main Body ◽  
Mutant Form ◽  
Exotoxin A ◽  
Elongation Factor 2 ◽  
Pseudomonas Aeruginosa Exotoxin

CRM66 (cross-reactive 66kDa protein) is an inactive mutant form of Pseudomonas aeruginosa exotoxin A that has been isolated from a mutant strain of P. aeruginosa derived from nitrosoguanidine-based mutagenesis. The mutation within this enzyme toxin was previously identified as H426Y and it was shown to possess significantly reduced enzymic activity. Furthermore, it was previously suggested that His-426 may directly participate in the catalytic mechanism of the exotoxin A enzyme and that it may also play an important role in the binding of the protein substrate of exotoxin A, a critical protein factor in eukaryotic protein translation known as elongation factor-2. In order to more thoroughly characterize the role of His-426 in the enzyme mechanism of exotoxin A, amino acid substitutions were made within helix 1 of the enzyme domain in the vicinity of the His-426 residue. Analysis of the site-directed mutagenesis results involving kinetic and protein structural integrity measurements revealed that His-426 H-bonds to Tyr-502 and that replacement of His-426 with polar substitutions leads to structural alterations of the enzyme's folded conformation. Furthermore, it was shown that His-426 is not important for the binding of either of the two substrates of exotoxin A, NAD+ or elongation factor-2. In summary, these data show that His-426 is not an active-site residue and that it is not important for substrate binding or orientation, but that it plays an important structural role in helping to maintain the folded conformation of the enzyme toxin. Therefore, the role of His-426 would seem to be to tether helix 1 to the main body of the enzyme, and mutations resulting in the disruption of this region of the enzyme result in a significantly impaired enzyme.

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