The early translocation of anthrax lethal factor by atomically detailed simulations and Milestoning theory

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Date

2021-08-06

Authors

Ma, Piao, Ph. D.

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The pathogenesis of the anthrax toxin requires the translocation of the lethal factor (LF) or the edema factor (EF) from the endosome to the cytosol though the anthrax channel. The channel is formed by protective antigen (PA) proteins. In this dissertation, we are interested in the translocation of LF through the anthrax channel. The unstructured N-terminal segment (LFN) of LF consists of 30 residues and is leading the translocation event. A critical barrier for the translocation across the channel is the ϕ clamp which consists of seven phenylalanine (F) residues in a ring. It is the narrowest part of the channel and is considered the dividing line between the endosome and the cytosol solutions.

We use atomically detailed simulations to study the translocation process. The solvated system (the channel and LF) contains about 680,000 atoms. Therefore, the computational costs of straightforward molecular dynamics simulations are significant. To facilitate the atomically detailed calculations we use the method of Milestoning. Milestoning simulate the overall process using short trajectories between interfaces called milestones. The information from the short trajectories, which are computed efficiently, is used to build a stochastic model for the entire process.

LFN modifies its protonation state depending on the pH. Positively charged LFN permeates more easily through the ϕ clamp compared to a less charged LFN. This preference is explained by the high concentration of negatively charged residues at the inner side of the anthrax channel. The electrostatic interactions provide the driving force for the permeation of LFN. In vivo systems, the positively charged state is supported by the low pH in the endosome. Once LFN passes the ϕ clamp, the N-terminal peptide chain is fully stretched and exerts a force on the rest of the protein. It assists the unfolding of the rest of the protein in preparation for a complete translocation. Mutations of the F427 residues at the ϕ clamp impact the translocation.

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