The long range goals of this project are to understand structure- function relationships of the ADP -ribosylating exotoxins at the level of 3-dimensional crystallographic structure. The work described is focused on diphtheria toxin (DT) and Pseudomonas aeruginosa exotoxin A (ETA), two related toxins that block protein synthesis by ADP-ribosylation of Elongation Factor-2 (EF- 2). With respect to DT, studies will be performed: (i) to characterize the active site, by combining site-directed mutagenesis with various biochemical and biophysical methods; (ii) to characterize the interaction of fragment A with EF-2 by new approaches, including the use of diphthamide synthesized by organic chemistry, and new assays for detecting interaction of EF-2 with active toxin fragments; (iii) to select toxin mutants that are defective in membrane-insertion and/or channel- formation at acidic pH, by means of a new positive selection procedure in E. coli; (iv) to explore a new method to study translocation of the enzymically active fragment A moiety of DT to the trans face of planar lipid bilayers; and (v) to determine the 3-dimensional structures of monomeric native DT, an enzymically active fragment (fragment A), and mutant forms of particular interest. With respect to ETA, studies are proposed which largely parallel those describe above for DT. Results will be interpreted in terms of a 3.0 A crystallographic structure of ETA determined recently, and an emerging 3-dimensional structure of DT-dimer at similar resolution. These studies are pertinent to a variety of problems in biomedical science, including mechanisms by which proteins penetrate membranes, routes to new vaccines, and mechanisms of directing toxic proteins to specific cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37AI022021-09
Application #
3481162
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1984-07-01
Project End
1993-04-30
Budget Start
1992-05-01
Budget End
1993-04-30
Support Year
9
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Harvard University
Department
Type
Schools of Medicine
DUNS #
082359691
City
Boston
State
MA
Country
United States
Zip Code
02115
Akkaladevi, Narahari; Mukherjee, Srayanta; Katayama, Hiroo et al. (2015) Following Natures Lead: On the Construction of Membrane-Inserted Toxins in Lipid Bilayer Nanodiscs. J Membr Biol 248:595-607
Jiang, Jiansen; Pentelute, Bradley L; Collier, R John et al. (2015) Atomic structure of anthrax protective antigen pore elucidates toxin translocation. Nature 521:545-9
Boland, Erin L; Van Dyken, Crystal M; Duckett, Rachel M et al. (2014) Structural complementation of the catalytic domain of pseudomonas exotoxin A. J Mol Biol 426:645-55
Sharma, Onkar; Collier, R John (2014) Polylysine-mediated translocation of the diphtheria toxin catalytic domain through the anthrax protective antigen pore. Biochemistry 53:6934-40
Akkaladevi, N; Hinton-Chollet, L; Katayama, H et al. (2013) Assembly of anthrax toxin pore: lethal-factor complexes into lipid nanodiscs. Protein Sci 22:492-501
McCluskey, Andrew J; Olive, Andrew J; Starnbach, Michael N et al. (2013) Targeting HER2-positive cancer cells with receptor-redirected anthrax protective antigen. Mol Oncol 7:440-51
Naik, Subhashchandra; Brock, Susan; Akkaladevi, Narahari et al. (2013) Monitoring the kinetics of the pH-driven transition of the anthrax toxin prepore to the pore by biolayer interferometry and surface plasmon resonance. Biochemistry 52:6335-47
McCluskey, Andrew J; Collier, R John (2013) Receptor-directed chimeric toxins created by sortase-mediated protein fusion. Mol Cancer Ther 12:2273-81
Gogol, E P; Akkaladevi, N; Szerszen, L et al. (2013) Three dimensional structure of the anthrax toxin translocon-lethal factor complex by cryo-electron microscopy. Protein Sci 22:586-94
Rodnin, Mykola V; Kyrychenko, Alexander; Kienker, Paul et al. (2011) Replacement of C-terminal histidines uncouples membrane insertion and translocation in diphtheria toxin T-domain. Biophys J 101:L41-3

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