Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 107, Issue 8, Pages 3453-3457Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1000100107
Keywords
electron microscopy; protective antigen; normal mode flexible fitting
Categories
Funding
- National Science Foundation [0445936, MCB-0744732]
- National Institutes of Health [5R37AI022021]
- Direct For Biological Sciences
- Div Of Molecular and Cellular Bioscience [0445936] Funding Source: National Science Foundation
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A major goal in understanding the pathogenesis of the anthrax bacillus is to determine how the protective antigen (PA) pore mediates translocation of the enzymatic components of anthrax toxin across membranes. To obtain structural insights into this mechanism, we constructed PA-pore membrane complexes and visualized them by using negative-stain electron microscopy. Two populations of PA pores were visualized in membranes, vesicle-inserted and nanodisc-inserted, allowing us to reconstruct two virtually identical PA-pore structures at 22-angstrom resolution. Reconstruction of a domain 4-truncated PA pore inserted into nanodiscs showed that this domain does not significantly influence pore structure. Normal mode flexible fitting of the x-ray crystallographic coordinates of the PA prepore indicated that a prominent flange observed within the pore lumen is formed by the convergence of mobile loops carrying Phe427, a residue known to catalyze protein translocation. Our results have identified the location of a crucial functional element of the PA pore and documented the value of combining nanodisc technology with electron microscopy to examine the structures of membrane-interactive proteins.
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