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A kinetic analysis of protein transport through the anthrax toxin channel.

Basilio D, Kienker PK, Briggs SW, Finkelstein A - J. Gen. Physiol. (2011)

Bottom Line: As expected, the translocation rate is slower with more than one LF(N) bound.We also present a simple electrodiffusion model of translocation in which LF(N) is represented as a charged rod that moves subject to both Brownian motion and an applied electric field.The cumulative distribution of first-passage times of the rod past the end of the channel displays S-shaped kinetics with a voltage dependence in agreement with experimental data.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY 10461, USA. dab2043@-med.cornell.edu

ABSTRACT
Anthrax toxin is composed of three proteins: a translocase heptameric channel, (PA(63))(7), formed from protective antigen (PA), which allows the other two proteins, lethal factor (LF) and edema factor (EF), to translocate across a host cell's endosomal membrane, disrupting cellular homeostasis. (PA(63))(7) incorporated into planar phospholipid bilayer membranes forms a channel capable of transporting LF and EF. Protein translocation through the channel can be driven by voltage on a timescale of seconds. A characteristic of the translocation of LF(N), the N-terminal 263 residues of LF, is its S-shaped kinetics. Because all of the translocation experiments reported in the literature have been performed with more than one LF(N) molecule bound to most of the channels, it is not clear whether the S-shaped kinetics are an intrinsic characteristic of translocation kinetics or are merely a consequence of the translocation in tandem of two or three LF(N)s. In this paper, we show both in macroscopic and single-channel experiments that even with only one LF(N) bound to the channel, the translocation kinetics are S shaped. As expected, the translocation rate is slower with more than one LF(N) bound. We also present a simple electrodiffusion model of translocation in which LF(N) is represented as a charged rod that moves subject to both Brownian motion and an applied electric field. The cumulative distribution of first-passage times of the rod past the end of the channel displays S-shaped kinetics with a voltage dependence in agreement with experimental data.

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Structure of the (PA63)7 channel. The figure on the left was created from the PA channel atomic model (Protein Data Bank accession no. 1V36) reported by Nguyen (2004). The parallel dashed lines indicate the thickness of the hydrophobic interior of the phospholipid bilayer. The figure on the right is a top view of this structure. The three black outlines are LFNs and are placed where they are presumed to bind to the mushroom cap of the channel (Melnyk et al., 2006). Note that even though there are seven binding sites in the cap, only three LFNs can be accommodated.
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fig1: Structure of the (PA63)7 channel. The figure on the left was created from the PA channel atomic model (Protein Data Bank accession no. 1V36) reported by Nguyen (2004). The parallel dashed lines indicate the thickness of the hydrophobic interior of the phospholipid bilayer. The figure on the right is a top view of this structure. The three black outlines are LFNs and are placed where they are presumed to bind to the mushroom cap of the channel (Melnyk et al., 2006). Note that even though there are seven binding sites in the cap, only three LFNs can be accommodated.

Mentions: The toxin produced by Bacillus anthracis, the causative agent of anthrax, consists of three separate monomeric proteins. Two of them, edema factor (EF; 89 kD) and lethal factor (LF; 90 kD), are enzymes that produce their toxic effects upon gaining access to the cytosol of the target cell. Access is gained through the agency of the third protein, protective antigen (PA; 83 kD), which provides a channel for their entry into the cytosol from an intracellular acidic vesicle compartment in which they find themselves after receptor-mediated endocytosis. (For a general review of anthrax toxin, see Young and Collier, 2007.) The channel formed by PA, (PA63)7, is a heptamer of the 63-kD fragment left after the cleavage of a 20-kD fragment from the N-terminal end of PA. (An octameric channel can also be formed [Kintzer et al., 2009].) This channel is mushroom shaped with a long (100 Å) 14-stranded β-barrel stem (Fig. 1; Benson et al., 1998; Nassi et al., 2002; Nguyen, 2004; Katayama et al., 2008); near the entry to the stem is a ring of phenylalanines dubbed the Φ clamp (Krantz et al., 2005). The seven binding sites for EF and LF reside in the mushroom cap, and up to three molecules of EF and/or LF can simultaneously occupy these sites (Cunningham et al., 2002; Mogridge et al., 2002; Pimental et al., 2004; Young and Collier, 2007). The ligand-binding site spans the intersection between two PA63 subunits; steric constraints restrict the number of ligands bound to three (Fig. 1). EF and LF can be driven by voltage and pH gradients through (PA63)7 channels reconstituted in planar phospholipid bilayer membranes (Zhang et al., 2004a,b; Krantz et al., 2005, 2006; Basilio et al., 2009; Finkelstein, 2009), although most of these experiments, as the ones described in this paper, were performed with LFN, the 263-residue N-terminal portion of LF that binds to the ligand-binding sites (Young and Collier, 2007).


A kinetic analysis of protein transport through the anthrax toxin channel.

Basilio D, Kienker PK, Briggs SW, Finkelstein A - J. Gen. Physiol. (2011)

Structure of the (PA63)7 channel. The figure on the left was created from the PA channel atomic model (Protein Data Bank accession no. 1V36) reported by Nguyen (2004). The parallel dashed lines indicate the thickness of the hydrophobic interior of the phospholipid bilayer. The figure on the right is a top view of this structure. The three black outlines are LFNs and are placed where they are presumed to bind to the mushroom cap of the channel (Melnyk et al., 2006). Note that even though there are seven binding sites in the cap, only three LFNs can be accommodated.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3105512&req=5

fig1: Structure of the (PA63)7 channel. The figure on the left was created from the PA channel atomic model (Protein Data Bank accession no. 1V36) reported by Nguyen (2004). The parallel dashed lines indicate the thickness of the hydrophobic interior of the phospholipid bilayer. The figure on the right is a top view of this structure. The three black outlines are LFNs and are placed where they are presumed to bind to the mushroom cap of the channel (Melnyk et al., 2006). Note that even though there are seven binding sites in the cap, only three LFNs can be accommodated.
Mentions: The toxin produced by Bacillus anthracis, the causative agent of anthrax, consists of three separate monomeric proteins. Two of them, edema factor (EF; 89 kD) and lethal factor (LF; 90 kD), are enzymes that produce their toxic effects upon gaining access to the cytosol of the target cell. Access is gained through the agency of the third protein, protective antigen (PA; 83 kD), which provides a channel for their entry into the cytosol from an intracellular acidic vesicle compartment in which they find themselves after receptor-mediated endocytosis. (For a general review of anthrax toxin, see Young and Collier, 2007.) The channel formed by PA, (PA63)7, is a heptamer of the 63-kD fragment left after the cleavage of a 20-kD fragment from the N-terminal end of PA. (An octameric channel can also be formed [Kintzer et al., 2009].) This channel is mushroom shaped with a long (100 Å) 14-stranded β-barrel stem (Fig. 1; Benson et al., 1998; Nassi et al., 2002; Nguyen, 2004; Katayama et al., 2008); near the entry to the stem is a ring of phenylalanines dubbed the Φ clamp (Krantz et al., 2005). The seven binding sites for EF and LF reside in the mushroom cap, and up to three molecules of EF and/or LF can simultaneously occupy these sites (Cunningham et al., 2002; Mogridge et al., 2002; Pimental et al., 2004; Young and Collier, 2007). The ligand-binding site spans the intersection between two PA63 subunits; steric constraints restrict the number of ligands bound to three (Fig. 1). EF and LF can be driven by voltage and pH gradients through (PA63)7 channels reconstituted in planar phospholipid bilayer membranes (Zhang et al., 2004a,b; Krantz et al., 2005, 2006; Basilio et al., 2009; Finkelstein, 2009), although most of these experiments, as the ones described in this paper, were performed with LFN, the 263-residue N-terminal portion of LF that binds to the ligand-binding sites (Young and Collier, 2007).

Bottom Line: As expected, the translocation rate is slower with more than one LF(N) bound.We also present a simple electrodiffusion model of translocation in which LF(N) is represented as a charged rod that moves subject to both Brownian motion and an applied electric field.The cumulative distribution of first-passage times of the rod past the end of the channel displays S-shaped kinetics with a voltage dependence in agreement with experimental data.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY 10461, USA. dab2043@-med.cornell.edu

ABSTRACT
Anthrax toxin is composed of three proteins: a translocase heptameric channel, (PA(63))(7), formed from protective antigen (PA), which allows the other two proteins, lethal factor (LF) and edema factor (EF), to translocate across a host cell's endosomal membrane, disrupting cellular homeostasis. (PA(63))(7) incorporated into planar phospholipid bilayer membranes forms a channel capable of transporting LF and EF. Protein translocation through the channel can be driven by voltage on a timescale of seconds. A characteristic of the translocation of LF(N), the N-terminal 263 residues of LF, is its S-shaped kinetics. Because all of the translocation experiments reported in the literature have been performed with more than one LF(N) molecule bound to most of the channels, it is not clear whether the S-shaped kinetics are an intrinsic characteristic of translocation kinetics or are merely a consequence of the translocation in tandem of two or three LF(N)s. In this paper, we show both in macroscopic and single-channel experiments that even with only one LF(N) bound to the channel, the translocation kinetics are S shaped. As expected, the translocation rate is slower with more than one LF(N) bound. We also present a simple electrodiffusion model of translocation in which LF(N) is represented as a charged rod that moves subject to both Brownian motion and an applied electric field. The cumulative distribution of first-passage times of the rod past the end of the channel displays S-shaped kinetics with a voltage dependence in agreement with experimental data.

Show MeSH
Related in: MedlinePlus