Peptide- and proton-driven allosteric clamps catalyze anthrax toxin translocation across membranes

Abstract

Anthrax toxin is an intracellularly acting toxin in which sufficient information is available regarding the structure of its transmembrane channel, allowing for detailed investigation of models of translocation. Anthrax toxin, comprising three proteins-protective antigen (PA), lethal factor (LF), and edema factor-translocates large proteins across membranes. Here we show that the PA translocase channel has a transport function in which its catalytic active sites operate allosterically. We find that the phenylalanine clamp (ϕ-clamp), the known conductance bottleneck in the PA translocase, gates as either a more closed state or a more dilated state. Thermodynamically, the two channel states have >300-fold different binding affinities for an LFderived peptide. The change in clamp thermodynamics requires distant α-clamp and ϕ-clamp sites. Clamp allostery and translocation are more optimal for LF peptides with uniform stereochemistry, where the least allosteric and least efficiently translocated peptide had a mixed stereochemistry. Overall, the kinetic results are in less agreement with an extended-chain Brownian ratchet model but, instead, are more consistent with an allosteric helix-compression model that is dependent also on substrate peptide coil-to-helix/ helix-to-coil cooperativity.allostery | planar bilayer electrophysiology | anthrax toxin | helix-compression model | Brownian ratchet

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

“…The helix-compression model, on the other hand, suggests that the polypeptide clamp sites are dynamic and under allosteric control [17]. During translocation, the translocating chain binds protons with its acidic groups and forms an α helix inside the channel.…”

Effect of the Endosomal Acidification on Small Ion Transport Through the Anthrax Toxin PA63 Channel

“…The alternate conductance states of open PA 63 channels were recently rediscovered by Das and Krantz [32] (see discussion in refs [37,38]). To provide support for the allosteric helix compression model of LF translocation through PA 63 [39], the authors hypothesize that the two conductance states of PA 63 represent the so-called 'clamped empty' and 'unclamped empty dilated' states of the channel, with the dilation occurring at the -clamp.…”

“…Therefore, if indeed the main to maximum state transformation represents PA 63 channel dilation at the -clamp region, as claimed by the ref. [32] authors, it is unclear why both AmPrCD and MTPP blockers are not sensitive to these structural changes. In addition, at pH > 5.5 PA 63 channels…”

“…In addition, if this effect does represent the dilation of the channel lumen at the -clamp region, important for the protein transport as suggested in ref. [32], it is unclear why the 'two-conductance-state' behavior is more prominent at pH  6.5 and high salt concentrations and why  33 % of the channels at pH 6.6 were found to insert into the maximum conductance state without any change-over to the main state [31]. Considering the fact that at neutral pH ( Fig.…”

“…Note that pH dependence of the PA 63 single-channel current has the following four distinctive features. (1) Not only do the two alternate conductance states appear at pH  5.5 [32], but the difference between the states becomes smaller with lowering pH from 7.5 to 6 and nearly vanishes at pH 5.5.…”