This project encompasses a range of structural studies on two secreted bacterial toxins, Escherichia coli heat labile enterotoxin (LT) and cholera toxin (CT). The severe diarrheal disease cause by cholera toxin may result in death within hours. The milder infectious diarrhea produced by LT is rarely life-threatening in the developed world, but is a major cause of infant death in the third world.
Specific aims of this proposal include x-ray crystallographic investigation of the fully active ADP-ribosylating A subunit to reveal catalytic mechanism and substrate binding modes, or engineered variants of the toxins, of the native toxins complexed with candidate inhibitors developed through structure-based drug design, and of hybrid molecular assemblies which use the native toxin structure as a scaffold for vaccine design. Long-term goals of this project are: (1) to investigate fundamental biological questions including the catalytic mechanism of ADP- ribosylation, structural determinants of toxin assembly, and the structural basis for recognition of complex sacharides. (2) to guide design of drugs effective against enterotoxigenic disease by providing a structural explanation for the biological function and activity of these toxins. LT and CT are 80% identical in sequence, exhibit similar subunit assembly, are immunologically cross-reactive, bind specifically to the same cell surface receptor, and share a common catalytic activity. Three aspects targeted for structure-based drug design are toxin assembly, receptor binding, and catalytic activity. (3) to use the remarkable ability of LT and CT to stimulate the mucosal immune system by designing prototype vaccines which retain the desirable immunological properties of the toxin while minimizing or abolishing the toxic activity. This work comprises structural study of engineered variants of the native toxins and of hybrid protein assemblies which incorporate foreign epitopes into the toxin structure.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI034501-10
Application #
6534034
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Hall, Robert H
Project Start
1993-09-01
Project End
2003-08-31
Budget Start
2002-09-01
Budget End
2003-08-31
Support Year
10
Fiscal Year
2002
Total Cost
$347,373
Indirect Cost
Name
University of Washington
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
135646524
City
Seattle
State
WA
Country
United States
Zip Code
98195
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