The Protein Translocase Activity of Anthrax Toxin Protective Antigen is Stereoselective

Abstract

Translocase channels deliver proteins across membranes within living cells. Many classes of these translocases have narrow pores that require substrate unfolding. Additionally, transport is known to be highly directional. These energy intensive processes of course require the consumption of an energy gradient. The conformation of the translocating chain is likely critical to these mechanisms; however, these areas are largely unexplored due in large part to the lack of appropriate biophysical models. Presently, we use anthrax toxin as a tractable model to study translocation. The toxin is comprised of a channel, called protective antigen (PA), which translocates its two substrates, lethal and edema factors (LF and EF, 90kDa each), across a host cell's endosomal membrane. Using planer lipid bilayer electrophysiology, we have demonstrated the translocation of full length LF, EF, and the N-terminal domain of LF (LFN : LF residues 1-263) via the PA oligomer. Translocation is driven by a membrane potential (Δψ) or proton gradient (ΔpH), albeit the latter is likely more physiological. Here we report single-channel and ensemble studies of translocation using long synthetic peptides designed in a manner that only their stereochemistry is varied. Specifically, we have taken the first 50 residues of LF and constructed peptides with all L-amino acids, all D-amino acids, and mixtures of D and L amino acids. The Δψ- or ΔpH-dependent translocation of these different peptides reveals that the peptides with uniform stereochemistry translocate more rapidly than those with mixed stereochemistry. These data are consistent with the model that the substrate does not solely translocate in an extended conformation, but rather the substrate also forms more-compact helical states only accessible to substrates with uniform stereochemistry.