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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.

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 edema factor (EF). Atx forms an active holotoxin complex containing a ring-shaped PA oligomer bound to multiple copies of LF and EF. 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 EF 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 catalyze 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.

Anthrax toxin (Atx) is a key virulence factor secreted by Bacillus anthracis. This three-protein toxin includes protective antigen (PA) and two enzymatic components, lethal factor (LF) and edema factor (EF), which must assemble into oligomeric complexes to disrupt cell physiology. Atx complexes are endocytosed, where they convert to a transmembrane channel that transports LF and EF into the cytosol. Assembly from monomeric components may occur in two physiological contexts: 1) in the bloodstream and 2) on cell surfaces. We have previously shown that the assembly of toxin complexes on cell surfaces produces a mixture of ring-shaped homooctameric or homoheptameric PA oligomers in a 1:2 ratio, which assemble via dimeric PA intermediates. Here we investigate how Atx complexes assemble in bovine blood. We find that, under these conditions, the predominant toxin complex that assembles is octameric. Incubation in blood causes heptameric LT complexes to lose greater than 90% cytotoxicity, whereas octameric LT complexes retain their activity. To understand the molecular mechanism of the apparent differential loss of toxin activity, we determined the pH-threshold of conversion to the channel state for each oligomeric complex. We find that the octameric toxin has a lower pH-threshold for channel formation than the heptamer. A consequence of this is that heptamers are inactivated by premature conversion to the channel state, whereas the octamers remain in the functional prechannel state. We propose that assembly of two oligomeric Atx complexes may provide a regulation mechanism for anthrax toxin cytotoxicity in both assembly at cell-surfaces and in the bloodstream. The assembly of octameric toxin complexes at the cell surface may alleviate potential assembly bottlenecks incurred by the mechanism of assembly via PA dimers, while in the bloodstream they may serve to maintain cytotoxicity during anthrax pathogenesis.

The reduction potential of the (FADH-/FADH*) couple in DNA photolyase was measured, and the value was found to be significantly higher than the values estimated in the literature. In the absence of substrate, the enzyme has a reduction potential of 16 +/- 6 mV vs NHE. In the presence of excess substrate the reduction potential increases to 81 +/- 8 mV vs NHE. The increase in reduction potential has physiological relevance since it gives the catalytic state greater resistance to oxidation. This is the first measurement of a reduction potential for this class of DNA-repair enzymes and the larger family of blue-light photoreceptors.