Determining the Essential Unfolding Step in Protein Translocation using Anthrax Toxin

Abstract

Protein translocation across membranes is crucial to many cellular processes, which includes bacterial pathogenesis. However, its exact molecular mechanism is not well understood. An established model to study this process is the anthrax toxin, a three-protein virulence factor that intoxicates cells via transmembrane protein translocation. Following endocytosis of the toxin, the protective antigen (PA) forms a transmembrane channel, triggered by the acidic endosomal pH, through which the two active enzyme components, the lethal factor (LF) and the edema factor (EF) are translocated to the cytosol of the host cell. Two key non-specific polypeptide binding sites – the α-clamp and Φ-clamp – catalyze protein translocation across the PA channel. The Φ-clamp is a very narrow opening, thus requiring proteins to unfold during translocation. We hypothesize that the PA channel can be used to translocate small heterologous proteins, such as Im7, and that translocation occurs by protein unfolding through a rate-limiting transition state. We expressed and purified small proteins composed of Im7 wild type attached to the first 30 amino acids of LF (LF1-30.Im7 WT), and its 10 single amino acid mutants. Planar lipid bilayer electrophysiology was used to perform single channel translocation assays of the WT and its mutants. Our results revealed that the mutants translocated faster and at lower voltages than the wild type, indicating protein destabilization due to mutation of the core residues. This destabilization facilitated the unfolding process, which reduced the driving force necessary for their translocation. Moreover, the critical step in translocation can be determined by mapping the transition states through denaturation studies using guanidine hydrochloride. Understanding the mechanism of protein unfolding can significantly contribute to the development of a new model for targeted drug delivery.