Pseudomonas aeruginosa exotoxin A interaction with eucaryotic elongation factor 2. Role of the His426 residue.
Pseudomonas aeruginosa exotoxin A (ETA) catalyzes the transfer of the ADP-ribose moiety of NAD+ onto eucaryotic elongation factor 2 (EF-2). To study the ETA site of interaction with EF-2, an immobilized EF-2 binding assay was developed. This assay demonstrates that ETA, in the presence of NAD+, binds to immobilized EF-2. Additionally, diphtheria toxin was also found to bind to the immobilized EF-2 in the presence of NAD+. Comparative analysis was performed with a mutated form of ETA (CRM 66) in which a histidine residue at position 426 has been replaced with a tyrosine residue. This immunologically cross-reactive, ADP-ribosyl transferase-deficient toxin does not bind to immobilized EF-2, thus explaining its lack of ADPRT activity. ETA bound to immobilized EF-2 cannot bind the monoclonal antibody TC-1 which specifically recognizes the ETA epitope containing His426. Immunoprecipitation of native ETA by mAb TC-1 is only achieved by incubating ETA in the presence of NAD+. Diethyl pyrocarbonate modification of the His426 residue blocks ETA binding to EF-2 and prevents the binding of the TC-1 antibody. Analogs of NAD+ containing a reduced nicotinamide ring or modified adenine moieties cannot substitute for NAD+ in the immobilized binding assay. Collectively, these data support our proposal that the site of ETA interaction with EF-2 includes His426 and that a molecule of NAD+ is required for stable interaction.
Kessler
S P
SP
Dept. of Microbiology, Ohio State University, Columbus 43210-1292.
Galloway
D R
DR
D036002:ADP Ribose Transferases; D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D004047:Diethyl Pyrocarbonate / Q000737:chemistry; D005098:Exotoxins / Q000737:chemistry* / Q000276:immunology; D006639:Histidine / Q000737:chemistry; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000737:chemistry*; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa; D013329:Structure-Activity Relationship; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D004047:Diethyl Pyrocarbonate; D005098:Exotoxins; D006639:Histidine; D009243:NAD; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D011065:Poly(ADP-ribose) Polymerases; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A