To function, a protein must be correctly localized in the cell, especially in ones that are internally compartmentalized by membrane bilayers. Proteinaceous, membrane-embedded transporters, called translocase channels, can traffic proteins across membranes by a process known as transmembrane protein translocation. Translocase channels also play key functional roles in microbial pathogenesis, because a host cell's lipid bilayer membrane functions as a formidable, first line of defense, isolating the pathogen from its cytosol. The bacterium, Bacillus anthracis, for example, secretes a three-protein toxin, called anthrax toxin, which is composed of protective antigen (PA), lethal factor (LF), and edema factor (EF). PA assembles into a translocase channel, forming a narrow passageway across the host cell's endosomal membrane bilayer, but the channel is so narrow that LF and EF traverse it as unfolded polypeptide chains. Once inside the target cell's cytosol, LF and EF refold and then catalyze reactions that disrupt the cell's normal physiology. Studies of protein unfolding and transmembrane translocation probe exciting biophysical questions, which apply broadly to the studies of soluble molecular motors, which unfold, disassemble, and degrade proteins. How is a stable substrate protein unfolded in the cell? What structural features in the translocase channel determine the complex energy landscape that guides a chemically-complex, unfolded chain through the narrow confines of the channel? The biophysical chemistry of transmembrane protein translocation, however, has been challenging to characterize, and the three- dimensional structures of nearly all translocase channels are unknown. Bacterial toxins, like anthrax toxin, are particularly well-suited for these studies, because they carry their own translocase-channel machinery, which is able to spontaneously insert into lipid bilayer membranes. We will couple the spectroscopic tools used to study how proteins fold and unfold with planar lipid bilayer electrophysiology. (1) We will analyze in detail the thermodynamic and kinetic mechanisms, which describe how the translocase channel of anthrax toxin unfolds its substrate proteins, exploring the role of chaperone-like, active-site surfaces in the PA channel. (2) We will dissect Brownian-ratchet translocation models through ensemble and single-channel electrophysiology studies of artificial, designed polypeptide substrates. (3) The structure and assembly of the PA channel will be pursued using spectroscopy, electrophysiology, electron microscopy, and crystallography. Relevance: Knowledge of protein translocation mechanisms are of practical importance not only to developing novel methods to neutralize the toxin but also to advancing technologies, which exploit toxins as delivery vehicles for heterologous antigens and cytotoxins into immune and cancer cells.
The scope of this application covers a structure/function study of the problem of cellular protein unfolding and transport. We will focus on anthrax toxin, a three-protein, bacterial toxin secreted by Bacillus anthracis. We are seeking to obtain a biophysical understanding of the toxin's transmembrane translocation mechanism, which allows its cytotoxic cargo to enter into mammalian host cells.
|Ghosal, Koyel; Colby, Jennifer M; Das, Debasis et al. (2017) Dynamic Phenylalanine Clamp Interactions Define Single-Channel Polypeptide Translocation through the Anthrax Toxin Protective Antigen Channel. J Mol Biol 429:900-910|
|Das, Debasis; Krantz, Bryan A (2017) Secondary Structure Preferences of the Anthrax Toxin Protective Antigen Translocase. J Mol Biol 429:753-762|
|Krantz, Bryan A (2017) Reply to Yamini and Nestorovich: Alternate clamped states of the anthrax toxin protective antigen channel. Proc Natl Acad Sci U S A 114:E2547|
|Biondi, Elisa; Lane, Joshua D; Das, Debasis et al. (2016) Laboratory evolution of artificially expanded DNA gives redesignable aptamers that target the toxic form of anthrax protective antigen. Nucleic Acids Res 44:9565-9577|
|Das, Debasis; Krantz, Bryan A (2016) Peptide- and proton-driven allosteric clamps catalyze anthrax toxin translocation across membranes. Proc Natl Acad Sci U S A 113:9611-6|
|Jorgensen, Ine; Zhang, Yue; Krantz, Bryan A et al. (2016) Pyroptosis triggers pore-induced intracellular traps (PITs) that capture bacteria and lead to their clearance by efferocytosis. J Exp Med 213:2113-28|
|Krantz, Bryan A (2016) Anthrax lethal toxin co-complexes are stabilized by contacts between adjacent lethal factors. J Gen Physiol 148:273-5|
|Colby, Jennifer M; Krantz, Bryan A (2015) Peptide Probes Reveal a Hydrophobic Steric Ratchet in the Anthrax Toxin Protective Antigen Translocase. J Mol Biol 427:3598-3606|
|Brown, Michael J; Thoren, Katie L; Krantz, Bryan A (2015) Role of the ? Clamp in the Protein Translocation Mechanism of Anthrax Toxin. J Mol Biol 427:3340-9|
|Abrami, Laurence; Brandi, Lucia; Moayeri, Mahtab et al. (2013) Hijacking multivesicular bodies enables long-term and exosome-mediated long-distance action of anthrax toxin. Cell Rep 5:986-96|
Showing the most recent 10 out of 25 publications