The role of the diphthamide-containing loop within eukaryotic elongation factor 2 in ADP-ribosylation by Pseudomonas aeruginosa exotoxin A.
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.
Zhang
Yong
Y
Department of Molecular and Cellular Biology, Biophysics Interdepartmental Group, University of Guelph, Guelph, ON, Canada.
Liu
Suya
S
Lajoie
Gilles
G
Merrill
A Rod
AR
D036002:ADP Ribose Transferases / Q000494:pharmacology*; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000494:pharmacology*; D054736:Elongation Factor 2 Kinase / Q000737:chemistry / Q000378:metabolism*; D005098:Exotoxins / Q000494:pharmacology*; D017353:Gene Deletion; D015966:Gene Expression Regulation, Fungal / Q000502:physiology; D006639:Histidine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D037521:Virulence Factors / Q000494:pharmacology*; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000246:Adenosine Diphosphate Ribose; D001427:Bacterial Toxins; D054736:Elongation Factor 2 Kinase; D005098:Exotoxins; D006639:Histidine; D029701:Saccharomyces cerevisiae Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C083761:DPH2 protein, S cerevisiae; C027527:diphthamide