Dynamic Phenylalanine Clamp Interactions Define Single-Channel Polypeptide Translocation through the Anthrax Toxin Protective Antigen Channel

Abstract

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 L-amino acids translocated more efficiently than peptides synthesized with D- or alternating D,L-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.

Probing Translocation in Mutants of the Anthrax Channel: Atomically Detailed Simulations with Milestoning

“…In Figure , we plot the forward and backward MFPTs of the two mutants and the WT channels. The time courses illustrate the significance of the ϕ-clamp as a critical bottleneck, in accord with recent experiments . The bottleneck can be effective in both forward and backward reactions.…”

“…The time courses illustrate the significance of the ϕ clamp as a critical bottleneck, in accord with recent experiments. 39 The bottleneck can be effective in both forward and backward reactions. For translocation efficiency, the permeating chain is not only motivated to go forward, but is also blocked from going backward.…”

The Impact of Protonation on Early Translocation of Anthrax Lethal Factor: Kinetics from Molecular Dynamics Simulations and Milestoning Theory

“…Protonation of the histidines produces an intermediate result (light-blue curve) in which a metastable state is still observed that is only slightly less stable than the reactants. Metastable states have been detected in a recent experiment on peptide permeation through the anthrax channel . The black curve was calculated with variable protonation conditions.…”

“…Metastable states have been detected in a recent experiment on peptide permeation through the anthrax channel. 16 The black curve was calculated with variable protonation conditions. For every milestone, we computed the pK a of the LF N residues using PROPKA3, 17 as implemented in PDB2PQR, 18 and sampled protonation states for configurations that initiate milestone trajectories at pH 5.…”

Site-Specific Labeling and 19F NMR Provide Direct Evidence for Dynamic Behavior of the Anthrax Toxin Pore ϕ-Clamp Structure

“…In addition, singlechannel recordings of the PA pore in the presence of helixfavoring peptides revealed at least two intermediate conductance states, aside from the open and closed states, again indicating that the ϕ-clamp is a dynamic structure and not static. 34 To allow for translocation of EF and LF through the narrow ϕ-clamp, there must be some dynamic flexibility in this structure, and our 19 F NMR data would support a more dynamic ϕ-clamp that is likely able to expand and contract to sterically accommodate all 20 amino acids and combinations of sequences translocating through the channel.…”