Early Translocation of Anthrax Lethal Factor: Kinetics from Molecular Dynamics Simulations and Milestoning Theory

Abstract

We report atomically detailed simulations of the early translocation events of the lethal factor (LF) through the anthrax channel. Anthrax is a serious infectious disease caused by the bacterium known as Bacillus anthracis. The anthrax toxin consists of three proteins: protective antigen (PA), LF and edema factor (EF). PA is cleaved by a furin-related protease to generate PA63. PA63 then oligomerizes and forms a prepore complex that competitively binds LF or EF. The complex is then endocytosed and a PA channel is formed under the acidic condition in the endosome. The PA channel translocates LF and EF into cytosol, resulting in the death of the cell. The overall delivery of LF from endosome to cytosol can be divided into two major steps: pore formation, and translocation. Many experiments were conducted to differentiate between factors that influence the delivery of LF. However, an atomically detailed understanding of the function of the LF translocation machinery is missing. Here we focus on the first steps of the translocation step. We describe atomically detailed molecular dynamics simulations of the translocation of the LF N-terminal segment (LFN) through the wild type and mutated PA channels. The kinetics and thermodynamic properties of the early events of the translocation process are computed within the Milestoning theory. We illustrate that the initial event is strongly influenced by the protonation states of the permeating LFN. At the limit of maximum protonation, the free energy profile of the LFN translocation suggests that the system operates like a ratchet. Several mutants of the channel illustrate that long-range electrostatic interactions provide the dominant driving force for the translocation, while mutants with variable sizes cause smaller effects on translocation. Our simulation results agree qualitatively with published experimental results.