The unfolding story of anthrax toxin translocation

Abstract

The essential cellular functions of secretion and protein degradation require a molecular machine to unfold and translocate proteins either across a membrane or into a proteolytic complex. Protein translocation is also critical for microbial pathogenesis, namely bacteria can use translocase channels to deliver toxic proteins into a target cell. Anthrax toxin (Atx), a key virulence factor secreted by Bacillus anthracis, provides a robust biophysical model to characterize transmembrane protein translocation. Atx is comprised of three proteins: the translocase component, protective antigen (PA) and two enzyme components, lethal factor (LF) and oedema factor (OF). Atx forms an active holotoxin complex containing a ring-shaped PA oligomer bound to multiple copies of LF and OF. These complexes are endocytosed into mammalian host cells, where PA forms a protein-conducting translocase channel. The proton motive force unfolds and translocates LF and OF through the channel. Recent structure and function studies have shown that LF unfolds during translocation in a force-dependent manner via a series of metastable intermediates. Polypeptide-binding clamps located throughout the PA channel catalyse substrate unfolding and translocation by stabilizing unfolding intermediates through the formation of a series of interactions with various chemical groups and α-helical structure presented by the unfolding polypeptide during translocation.

Figure 1. Protein translocation challenges

A. A possible scheme for Atx assembly and entry into host cells. Proteolytically-activated PA monomers oligomerize into ring-shaped heptameric or octameric pre-channels, which can bind 3 or 4 LF/EF substrates, respectively. These complexes are endocytosed. Upon acidification of the endosome, the PA pre-channel converts to the channel state, ultimately allowing LF and EF to translocate into the cytosol. B. A possible protein unfolding and translocation pathway for Atx depicted in three successive steps: docking, protein unfolding, and translocation of the unfolded chain. C. Challenges encountered during translocation. During translocation substrates are mechanically unfolded by the driving force; the mechanical resistance, however, can vary significantly depending on the relative orientation of the substrate to the force vector. Combinatorial chemical complexity arises as the unfolded chain presents a wide array of changing combinations of side chain chemistries to the channel. Conformational heterogeneity is also present in the unfolded substrate polypeptides. Combinatorial chemical complexity and conformational heterogeneity present significant challenges for substrate recognition. Finally, counterproductive diffusive forces must also be overcome during translocation.

Figure 2. Molecular solutions to the challenges of protein translocation

Multiple clamping sites in the PA channel facilitate substrate unfolding by stabilizing partially unfolded intermediates. The α clamp elegantly mitigates the combinatorial chemical complexity and conformational heterogeneity of the substrate by recognizing uniformly shaped helical structures with broad sequence specificity (Feld et al., 2010). The α-clamp panel was rendered by morphing the protein databank (PDB) coordinates, 3KWV (Feld et al., 2010), with a model of the folded form of LF_N docked on PA using the PDB coordinates, 1J7N (Pannifer et al., 2001). The aromatic residues in the Φ clamp also provide a means to interact with many types of substrate chemistries by taking advantage of hydrophobic effect and allowing for broad specificity (Krantz et al., 2005). The ΔpH driving force can harness diffusive Brownian motion by changing the protonation state of anionic residues in the substrate during translocation (Krantz et al., 2006).