Anthrax is an ancient disease that has become a renewed concern owing to its threatened use as a weapon of biological warfare and terrorism. Lethal Toxin is the two-part protein toxin secreted by Bacillus anthracis, the causative agent of Anthrax, which is sufficient for death in experimental animals. The purpose of this work is to determine the atomic-level mechanisms of intoxication by Lethal Toxin, which comprises the Protective Antigen (PA, 83 kDa) and the Lethal Factor (LF, 90 kDa). These studies will contribute directly to the development of novel therapeutic agents, both in combating the disease of anthrax itself, and in the field of """"""""targeted toxins."""""""" We recently published the crystal structure of PA in its monomeric and water-soluble heptameric forms (PA63). Based on its structure, we have formulated a specific hypothesis of pH-induced conformational change leading to the creation of a membrane-spanning beta-barrel. We will test this hypothesis by determining the crystal structure of PA63 in its membrane-bound conformation. PA63 is the central component of a protein translocation machine that delivers LF into the host cell cytosol. In vitro, LF in combination with PA specifically kills macrophages, although it is able to enter all cell types tested. Recent work has shown that LF is a metalloprotease, and that one of its targets is MAP kinase kinase, although it is not clear yet how this activity is related to its pathogenicity. In order to shed light on the catalytic basis of LF action and its regulation, we will determine its atomic resolution structure and complexes with peptide fragments of its target proteins. These crystal structures will pave the way for studying the 7:7 translocation complex between the PA63 heptamer and Lethal Factor, which will provide testable hypotheses of the translocation process. Although these complexes are very large, our atomic-resolution determinations of the constituent elements, together with the 7-fold symmetry, will allow us to build reliable models even with limited resolution data. The work will complement the biochemical and biophysical experiments of our collaborators, Drs. Stephen Leppla at NIH Bethesda, Philip Hanna at Duke University, R. John Collier at Harvard Medical School and Alok Mitra at Scripps.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI047152-05
Application #
6743160
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Baker, Phillip J
Project Start
2000-04-01
Project End
2005-03-31
Budget Start
2004-04-01
Budget End
2005-03-31
Support Year
5
Fiscal Year
2004
Total Cost
$341,250
Indirect Cost
Name
Sanford-Burnham Medical Research Institute
Department
Type
DUNS #
020520466
City
La Jolla
State
CA
Country
United States
Zip Code
92037