Dynamic Phenylalanine Clamp Interactions Define Single-Channel Polypeptide Translocation through the Anthrax Toxin Protective Antigen Channel. Issue 6 (24th March 2017)

• Record Type: Journal Article
• Authors: Ghosal, Koyel; Colby, Jennifer M.; Das, Debasis; Joy, Stephen T.; Arora, Paramjit S.; Krantz, Bryan A.
• Is Part Of: Journal of molecular biology. Volume 429:Issue 6(2017)
• Page Start: 900
• Page End: 910
• Publication Date: 2017-03-24
• Subjects: PA protective antigen -- LF lethal factor -- EF edema factor -- Δψ membrane potential -- α clamp α helix binding clamp -- ϕ clamp phenylalanine clamp -- CDF cumulative distribution function -- WT wild-type -- HBS hydrogen bond surrogate -- UBB universal bilayer buffer; Bacillus anthracis -- anthrax toxin -- protective antigen -- electrophysiology -- peptide clamp; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2017.02.005
• Languages: English
• ISSNs: 0022-2836

Abstract

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 ʟ-amino acids translocated more efficiently than peptides synthesized withD - or alternatingD, 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. Graphical Abstract: Highlights: Small guest/host peptide probes were tested on protective antigen channels. Tryptophan containing peptides translocated while oscillating between intermediates. The oscillating intermediates were part of the translocation pathway. Prenucleated helix peptide translocated poorly through wild-type channels. Efficient translocation requires peptide flexibility and dynamic phenylalanine clamp site.