title: Structural complementation of the catalytic domain of pseudomonas exotoxin A.
abstract: The catalytic moiety of Pseudomonas exotoxin A (domain III or PE3) inhibits protein synthesis by ADP-ribosylation of eukaryotic elongation factor 2. PE3 is widely used as a cytocidal payload in receptor-targeted protein toxin conjugates. We have designed and characterized catalytically inactive fragments of PE3 that are capable of structural complementation. We dissected PE3 at an extended loop and fused each fragment to one subunit of a heterospecific coiled coil. In vitro ADP-ribosylation and protein translation assays demonstrate that the resulting fusions-supplied exogenously as genetic elements or purified protein fragments-had no significant catalytic activity or effect on protein synthesis individually but, in combination, catalyzed the ADP-ribosylation of eukaryotic elongation factor 2 and inhibited protein synthesis. Although complementing PE3 fragments are catalytically less efficient than intact PE3 in cell-free systems, co-expression in live cells transfected with transgenes encoding the toxin fusions inhibits protein synthesis and causes cell death comparably as intact PE3. Complementation of split PE3 offers a direct extension of the immunotoxin approach to generate bispecific agents that may be useful to target complex phenotypes.
authors.affiliation: Department of Pharmaceutical Sciences, Washington State University, Pullman, WA 99164, USA.
authors.forename: Erin L
authors.initials: EL
authors.lastname: Boland
authors.affiliation: Department of Pharmaceutical Sciences, Washington State University, Pullman, WA 99164, USA.
authors.forename: Crystal M
authors.initials: CM
authors.lastname: Van Dyken
authors.affiliation: Department of Pharmaceutical Sciences, Washington State University, Pullman, WA 99164, USA.
authors.forename: Rachel M
authors.initials: RM
authors.lastname: Duckett
authors.affiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA. Electronic address: andrew_mccluskey@hms.harvard.edu.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: McCluskey
authors.affiliation: Department of Pharmaceutical Sciences, Washington State University, Pullman, WA 99164, USA. Electronic address: gpoon@wsu.edu.
authors.forename: Gregory M K
authors.initials: GM
authors.lastname: Poon
mesh_terms: D036002:ADP Ribose Transferases / Q000737:chemistry* / Q000378:metabolism*; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry* / Q000378:metabolism*; D020134:Catalytic Domain; D002470:Cell Survival; D002474:Cell-Free System; D002850:Chromatography, Gel; D005098:Exotoxins / Q000737:chemistry* / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D014176:Protein Biosynthesis; D037521:Virulence Factors / Q000737:chemistry* / Q000378:metabolism*; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D000246:Adenosine Diphosphate Ribose; D001427:Bacterial Toxins; D005098:Exotoxins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
keywords: ADP-ribosylation; ADP-ribosyltransferase; ADPRT; DT; MBP; RRL; SEM; cytotoxicity; diphtheria toxin; eEF2; elongation factor 2; eukaryotic elongation factor 2; exotoxin A; maltose-binding protein; rabbit reticulocyte lysate; standard error of the mean; structural complementation