$^{19}F$-NMR studies to understand pH induced structural changes in protective antigen anthrax toxin

Abstract

Anthrax bacteria ($Bacillus$ $anthracis$) produces Protective antigen toxin (PA), Lethal factor (LF) and Edema factor (EF) protein components into the host cells. Protective antigen toxin (PA83 - 83kDa polypeptide) binds to cell surface receptors. PA is cleaved into a 20kDa fragment $PA_{20}$ and 63kDa fragment $PA_{63}$ by Furin like protease. The $PA_{20}$ is released, resulting in spontaneous oligomerization into heptamer $(PA63)_{7}$ that facilitates EF and LF to bind. The complex is endocytosed and acidic conditions in endosome triggers conformational changes in prepore complex and leading to a membrane spanning pore formation. Formation of pore allows translocation of LF and EF components into the cell. In pore structure "O"-ring shaped narrow 6 Å diameter ring Φ-clamp is formed by seven phenylalanine residues (Phe427). The Φ-clamp is not wide enough to allow protein secondary structure elements, it may allow only fully unfolded polypeptides to pass through PA pore. To use $^{19}F$ NMR as a tool, PA83 is labelled site specifically with $p$-Fluoro-Phenyalalanine at Phe427 residue. Site specifically introduced $^{19}F$ probe helps to follow conformational changes and dynamic behavior of the Φ-clamp in prepore, $LF_{N}$ bound prepore and pore state. Our results indicate that $p$F-Phe427 is dynamic in the prepore state and then becomes more dynamic in the transition to the pore state. Further increase in dynamic behavior at the Φ-clamp in pore state may provide the necessary room for movement needed in translocating EF and LF into the cell cytosol.