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Anthrax toxin is an intracellularly acting toxin where sufficient detail is known about the structure of its channel, allowing for molecular investigations of translocation. The toxin is composed of three proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF). The toxin's translocon, PA, translocates the large enzymes, LF and EF, across the endosomal membrane into the host cell's cytosol. Polypeptide clamps located throughout the PA channel catalyze the translocation of LF and EF. Here, we show that the central peptide clamp, the ϕ clamp, is a dynamic site that governs the overall peptide translocation pathway. Single-channel translocations of a 10-residue, guest–host peptide revealed that there were four states when peptide interacted with the channel. Two of the states had intermediate conductances of 10% and 50% of full conductance. With aromatic guest–host peptides, the 50% conducting intermediate oscillated with the fully blocked state. A Trp guest–host peptide was studied by manipulating its stereochemistry and prenucleating helix formation with a covalent linkage in the place of a hydrogen bond or hydrogen-bond surrogate (HBS). The Trp peptide synthesized with ʟ-amino acids translocated more efficiently than peptides synthesized with D - or alternating D ,ʟ-amino acids. HBS stapled Trp peptide exhibited signs of steric hindrance and difficulty translocating. However, when mutant ϕ clamp (F427A) channels were tested, the HBS peptide translocated normally. Overall, peptide translocation is defined by dynamic interactions between the peptide and ϕ clamp. These dynamics require conformational flexibility, such that the peptide productively forms both extended-chain and helical states during translocation. [ABSTRACT FROM AUTHOR]

In order for many proteins to move across hydrophobic membrane bilayers, they must be unfolded and translocated by a membrane-embedded channel. These translocase channels interact with the substrate proteins they translocate via hydrophobic pore loops and cleft structures called clamps. The molecular basis for how clamps facilitate unfolding and translocation is poorly understood. Anthrax toxin is composed of three proteins, a translocase channel-forming subunit, called protective antigen (PA), and two substrate proteins, called lethal factor (LF) and edema factor. Oligomeric PA forms a large channel that contains three types of polypeptide clamp sites: an α clamp, a phenylalanine clamp, and a charge clamp. Currently, it is thought that these clamp sites operate allosterically and promote translocation via an allosteric helix compression mechanism. Here, we report on the substrate secondary structure dependence of the PA channel. Peptides derived from regions of LF with high α-helical content bound cooperatively, but those derived from β-sheet regions in LF did not, suggesting that an allosteric site preferentially recognizes α-helical structure over β-sheet structure. Peptides derived from helical sites in LF showed increasingly longer single-channel blockades as a function of peptide concentration, a result that was consistent with stronger clamping behavior and reduced backsliding. Moreover, peptides derived from helical regions of LF translocated more efficiently than peptides derived from β-sheet regions of LF. Overall, in support of the allosteric helix compression model, we find that the channel prefers α-helical sequences over β-sheet sequences. [ABSTRACT FROM AUTHOR]

Anthrax toxin is a tripartite virulence factor produced by Bacillus anthracis during infection. Under acidic endosomal pH conditions, the toxin's protective antigen (PA) component forms a transmembrane channel in host cells. The PA channel then translocates its two enzyme components, lethal factor and edema factor, into the host cytosol under the proton motive force. Protein translocation under a proton motive force is catalyzed by a series of nonspecific polypeptide binding sites, called clamps. A 10-residue guest/host peptide model system, KKKKKXXSXX, was used to functionally probe polypeptide-clamp interactions within wild-type PA channels. The guest residues were Thr, Ala, Leu, Phe, Tyr, and Trp. In steady-state translocation experiments, the channel blocked most tightly with peptides that had increasing amounts of nonpolar surface area. Cooperative peptide binding was observed in the Trp-containing peptide sequence but not the other tested sequences. Trp substitutions into a flexible, uncharged linker between the lethal factor amino-terminal domain and diphtheria toxin A chain expedited translocation. Therefore, peptide-clamp sites in translocase channels can sense large steric features (like tryptophan) in peptides, and while these steric interactions may make a peptide translocate poorly, in the context of folded domains, they can make the protein translocate more rapidly presumably via a hydrophobic steric ratchet mechanism. [ABSTRACT FROM AUTHOR]

Membrane-embedded molecular machines are utilized to move water-soluble proteins across these barriers. Anthrax toxin forms one such machine through the self-assembly of its three component proteins—protective antigen (PA), lethal factor, and edema factor. Upon endocytosis into host cells, acidification of the endosome induces PA to form a membrane-inserted channel, which unfolds lethal factor and edema factor and translocates them into the host cytosol. Translocation is driven by the proton motive force, composed of the chemical potential, the proton gradient (ΔpH), and the membrane potential (Δψ). A crystal structure of the lethal toxin core complex revealed an “α clamp” structure that binds to substrate helices nonspecifically. Here, we test the hypothesis that, through the recognition of unfolding helical structure, the α clamp can accelerate the rate of translocation. We produced a synthetic PA mutant in which an α helix was crosslinked into the α clamp to block its function. This synthetic construct impairs translocation by raising a yet uncharacterized translocation barrier shown to be much less force dependent than the known unfolding barrier. We also report that the α clamp more stably binds substrates that can form helices than those, such as polyproline, that cannot. Hence, the α clamp recognizes substrates by a general shape-complementarity mechanism. Substrates that are incapable of forming compact secondary structure (due to the introduction of a polyproline track) are severely deficient for translocation. Therefore, the α clamp and its recognition of helical structure in the translocating substrate play key roles in the molecular mechanism of protein translocation. [ABSTRACT FROM AUTHOR]