UC Berkeley

A fundamental problem in biochemistry is how molecular machines convert chemical potential energy into mechanical work. Here, this problem is addressed in terms of how a proton gradient (ΔpH) drives anthrax toxin transmembrane protein translocation with a given directionality. One model to explain this phenomenon is a charge-state Brownian ratchet, wherein movement is biased by asymmetries across the membrane. Acidic residues in the substrate are protonated on the lower pH starting side, thus allowing them to pass through the cation-selective protective antigen (PA) channel. Movement can occur in either direction according to Brownian motion, but acidic residues that reach the higher pH of the destination are deprotonated, preventing retrotranslocation and resolving movement in one direction.

This model is probed through the use of planar lipid bilayer electrophysiology to analyze the charge requirements of the model substrate LF_N, the binding domain of the natural substrate lethal factor. Acidic residues are necessary and sufficient for ΔpH-driven translocation provided the starting side pH is low enough to sufficiently protonate the residues. Their position in the sequence, just before LF_N's folded domain, plays a key role in substrate unfolding. Basic charges are important for initiation into the channel and to chaperone deprotonated acidic residues during translocation.

Further work by Sarah Wynia-Smith confirms the importance of acidic residues just before LF_N's folded domain and points to the existence of electrostatic translocation barriers in the PA channel. I identify key residues in the upper portion of the channel's β barrel that contribute to its cation selectivity. These residues also prove to play a significant role in substrate initiation and translocation, supporting the role for a charge gate in the channel that prevents retrotranslocation.

Finally, secondary structure of the translocating substrate is analyzed. LF_N translocates in a compact helical state nucleated by the channel's α clamp, a nonspecific helix-binding site recently identified by Geoffrey Feld. α clamp-induced helix formation lowers a previously identified but uncharacterized translocation barrier, and substrates that cannot form helices cannot translocate. This suggests a mechanism wherein helix melting, coupled to the electrostatic ratchet, drives translocation-coupled substrate unfolding.