Abstract
Anthrax lethal toxin is used as a model system to study protein translocation. The toxin is composed of a translocase channel, called protective antigen (PA), and an enzyme, called lethal factor (LF). A proton gradient (ΔpH) can drive LF unfolding and translocation through PA channels; however, the mechanism of ΔpH-mediated force generation, substrate unfolding, and establishment of directionality are poorly understood. One recent hypothesis suggests that the ΔpH may act through changes in the protonation state of residues in the substrate. Here we report the charge requirements of LF's amino-terminal binding domain (LF(N)) using planar lipid bilayer electrophysiology. We found that acidic residues are required in LF(N) to utilize a proton gradient for translocation. Constructs lacking negative charges in the unstructured presequence of LF(N) translocate independently of the ΔpH driving force. Acidic residues markedly increase the rate of ΔpH-driven translocation, and the presequence is optimized in its natural acidic residue content for efficient ΔpH-driven unfolding and translocation. We discuss a ΔpH-driven charge state Brownian ratchet mechanism for translocation, where glutamic and aspartic acid residues in the substrate are the "molecular teeth" of the ratchet. Our Brownian ratchet model includes a mechanism for unfolding and a novel role for positive charges, which we propose chaperone negative charges through the PA channel during ΔpH translocation.
Highlights
Transmembrane protein translocation [1,2,3,4] and intracellular protein degradation [5] are essential processes that allow the cell to traffic protein, form new organelles, maintain protein quality control, and regulate the cell cycle
Excess LF’s amino-terminal binding domain (LFN) in the cis compartment was removed by perfusion, and the translocation process was initiated by either changing the ⌬ and/or ⌬pH
Two types of parameters can be obtained from translocation records: the time for half of the protein to translocate (t1⁄2, measured in seconds) and the efficiency of translocation, which is equivalent to the fraction of LFN that successfully translocates
Summary
Constructs and Proteins—Site-directed mutagenesis was performed using a QuikChange procedure. The synthetic LFN constructs were overexpressed and purified, and their His tags were subsequently removed as described above (when required). Cis (side to which the PA oligomer is added) and trans chambers were bathed in the indicated buffers as required. Translocation Assays—Translocation experiments were carried out as described [6, 12] generally using a universal pH bilayer buffer system (UBB: 10 mM oxalic acid, 10 mM phosphoric acid, 10 mM MES, 1 mM EDTA). For translocations requiring a pH Ͼ 7.5, we used an altered UBB (6 mM oxalic acid, 6 mM phosphoric acid, 6 mM MES, 6 mM boric acid, 6 mM TAPS, 1 mM EDTA), which is better at buffering in the 7.5–9 range We found that these two types of buffers produced consistent translocation results. WT or a mutant LFN was added to the cis-side of the membrane, and a decrease in current was observed. Histograms of the current versus time data were fit to a two or three Gaussian function to obtain the relative percentages of time spent in the open, blocked, and partly blocked states
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