Anthrax: Every rose has its thorn

Figure 1. Fully assembled anthrax toxin ready for attack

Letters containing anthrax spores were mailed to several news media offices and two Democratic U.S. Senators, killing five people and infecting 17 others. The ensuing investigation became "one of the largest and most complex in the history of law enforcement.¹ Anthrax is well known for its thorn than its structural beauty which is often underappreciated due to its infamous connection with biological terrorism. 

Figure 2. Anthrax toxin side view

Anthrax toxin is a three-protein virulence factor secreted by Bacillus anthracis, and is composed of two A components (also known as lethal factor-LF, a cytotoxic enzyme), one B component (protective antigen-PA), and edema factor (EF). It is an example of a transmembrane protein-delivery system. The toxic complexes form lethal toxin (LT) that consists of protective antigen and lethal factor. Endocytosis of this LT complex causes the protein to unfold and PA region of the toxin to form a B-barrel so that lethal factor can be translocated into the cell similar to a virus inserting its genome into the host cell. The translocated LF then disrupts normal cellular physiology by cleaving mitogen-activated kinases. The mechanism of protein transport in mammalian systems require auxiliary proteins such as ATPases to provide energy to drive translocation. Similar mechanism is employed by the mechanism of protein transport through the anthrax channel which is a self-sufficient protein-translocating machine.²

Figure 3. Structural basis for anthrax lethal factor unfolding

Interestingly, unfolding of this protein is central for translocation into the host cell. Because folded proteins are thermodynamically stable under normal physiological conditions, the unfolding process requires complex and energy-consuming molecular mechanisms. The structural basis for the unfolding of anthrax lethal factor involves dimeric protective antigen oligomers. This structure contains a PA octamer bound to four LF PA-binding domains. The first α-helix and β-strand of each LF(N) unfold and dock into a deep amphipathic cleft on the surface of the PA octamer, which is called the α clamp. The α clamp possesses nonspecific polypeptide binding activity and is functionally relevant to efficient holotoxin assembly, PA.³ This crystal structure of this toxin was obtained and explored to provide insight into the mechanism of translocation-coupled protein unfolding. Though beautiful, each of the subunits seen in the complete assembly of the toxin dissociates and unfolds to burrow their way inside the cell. Like Angel hair in Christmas ornaments, anthrax toxin is beautiful to look at but one should not be in contact with it.

1. http://www.fbi.gov/anthrax/amerithraxlinks.htm

2. Blaustein, O. Robert. The anthrax toxin channel: a barrel of LFs. The Journal of Cell Biology, 2011.

3. Feld, Geoffrey,  Katie Thoren, Alexander, Kintzer, et al. Structural basis for the unfolding of anthrax lethal factor by protective antigen oligomers. Nature Structural & Molecular Biology 17(11): 1383-1391, 2010.

Assignment 2

Feld, Geoffrey,  Katie Thoren, Alexander, Kintzer, et al. Structural basis for the unfolding of anthrax lethal factor by protective antigen oligomers. Nature Structural & Molecular Biology 17(11): 1383-1391, 2010.

Anthrax toxin, a three-protein virulence factor secreted by Bacillus anthracis, is composed of two A components (also known as lethal factor-LF, a cytotoxic enzyme), one B component (protective antigen-PA), and edema factor (EF). It is an example of a transmembrane protein-delivery system. The toxic complexes form lethal toxin (LT) that consists of protective antigen and lethal factor. Endocytosis of this LT complex causes the protein to unfold and PA region of the toxin to form a B-barrel so that lethal factor can be translocated into the cell. The translocated LF then disrupts normal cellular physiology by cleaving mitogen-activated kinases. Interestingly, unfolding of this protein is central for translocation into the host cell. Because folded proteins are thermodynamically stable under normal physiological conditions, the unfolding process requires complex and energy-consuming molecular machinisms. The crystal structure of this toxin was obtained and explored to provide insight into the mechanism of translocation-coupled protein unfolding. Though beautiful, each of the subunits seen in the complete assembly of the toxin dissociates and unfolds to make their way inside the cell.

D., Basilio, Juris S.J., Collier R.J., Finkelstein A.  Evidence for a proton-protein symport mechanism in the anthrax toxin channel. Journal of General Physiology 133(3): 307-314, 2009.

                We know that PA incorporated into planar phospholipid bilayer membranes forms a B-barrel channel that transports LF and EF into the cell. This process is driven by a proton electrochemical potential gradient by a cis-positive voltage. What seemed paradoxical initially was that the N-terminal 263 residues of LF has a net negative charge and the channel strongly disfavors entry of such residues. The  translocation take place by neutralizing this positive charge by protonation. Aspartates and glutamates on the N-terminus of LF becomes neutralized and the translocated species exhibits overall positive charge. The driving force of translocation comes from the gradient of the electrochemical potential of protons but the overall process is more complicated than acid-induced unfolding of the toxin as it also requires electrophoresis through a passive pore. LF bears a net negative charge of -7 at pH 5.5 and -12 at pH 6.5, a symmetric pH at which translocation can be driven by voltage. Thus, LF_N bears a net negative charge at at least some of the pHs at which its translocation has been studied when the average pKs of the aspartates and glutamates on LF_N are shifted 1.5 units or more above their free amino acid values.

Blaustein, O. Robert. The anthrax toxin channel: a barrel of LFs. The Journal of Cell Biology, 2011.

                Pathogenesis of anthrax relies on receptor-mediated endocytosis of the toxin and vesicular acidification, pore formation in the endosomal membrane my PA-derived oligomer, and eventual translocation of EF and LF through the pore into the cytosol. Once inside, LF acts as a Zn-dependent protease that disrupts cellular signaling by cleaving proteins of the mitogen activated protein kinase family. Anthrax is the only A/B toxin for which translocation has been demonstrated convincingly due to its several unique features. For most A/B toxins, the A and B domains are part of a single protein which allows channel formation and translocation to occur simultaneously. Anthras toxin synthesizes PA, LF, and EF de novo as individual proteins and so the channel formation and translocation can be readily dissociated.

                The mechanism of protein transport in mammalian systems require auxiliary proteins such as ATPases to provide energy to drive translocation. Similar mechanism is employed by the mechanism of protein transport through the anthrax channel which is a self-sufficient protein-translocating machine. The author commented, “this is secaa barrel of LFs, I tell ya”.

Assignment 2

Feld, Geoffrey,  Katie Thoren, Alexander, Kintzer, et al. Structural basis for the unfolding of anthrax lethal factor by protective antigen oligomers. Nature Structural & Molecular Biology 17(11): 1383-1391, 2010.

Anthrax toxin, a three-protein virulence factor secreted by Bacillus anthracis, is composed of two A components (also known as lethal factor-LF, a cytotoxic enzyme), one B component (protective antigen-PA), and edema factor (EF). It is an example of a transmembrane protein-delivery system. The toxic complexes form lethal toxin (LT) that consists of protective antigen and lethal factor. Endocytosis of this LT complex causes the protein to unfold and PA region of the toxin to form a B-barrel so that lethal factor can be translocated into the cell. The translocated LF then disrupts normal cellular physiology by cleaving mitogen-activated kinases. Interestingly, unfolding of this protein is central for translocation into the host cell. Because folded proteins are thermodynamically stable under normal physiological conditions, the unfolding process requires complex and energy-consuming molecular machinisms. The crystal structure of this toxin was obtained and explored to provide insight into the mechanism of translocation-coupled protein unfolding. Though beautiful, each of the subunits seen in the complete assembly of the toxin dissociates and unfolds to make their way inside the cell.

D., Basilio, Juris S.J., Collier R.J., Finkelstein A.  Evidence for a proton-protein symport mechanism in the anthrax toxin channel. Journal of General Physiology 133(3): 307-314, 2009.

                We know that PA incorporated into planar phospholipid bilayer membranes forms a B-barrel channel that transports LF and EF into the cell. This process is driven by a proton electrochemical potential gradient by a cis-positive voltage. What seemed paradoxical initially was that the N-terminal 263 residues of LF has a net negative charge and the channel strongly disfavors entry of such residues. The  translocation take place by neutralizing this positive charge by protonation. Aspartates and glutamates on the N-terminus of LF becomes neutralized and the translocated species exhibits overall positive charge. The driving force of translocation comes from the gradient of the electrochemical potential of protons but the overall process is more complicated than acid-induced unfolding of the toxin as it also requires electrophoresis through a passive pore. LF bears a net negative charge of -7 at pH 5.5 and -12 at pH 6.5, a symmetric pH at which translocation can be driven by voltage. Thus, LF_N bears a net negative charge at at least some of the pHs at which its translocation has been studied when the average pKs of the aspartates and glutamates on LF_N are shifted 1.5 units or more above their free amino acid values.

Blaustein, O. Robert. The anthrax toxin channel: a barrel of LFs. The Journal of Cell Biology, 2011.

                Pathogenesis of anthrax relies on receptor-mediated endocytosis of the toxin and vesicular acidification, pore formation in the endosomal membrane my PA-derived oligomer, and eventual translocation of EF and LF through the pore into the cytosol. Once inside, LF acts as a Zn-dependent protease that disrupts cellular signaling by cleaving proteins of the mitogen activated protein kinase family. Anthrax is the only A/B toxin for which translocation has been demonstrated convincingly due to its several unique features. For most A/B toxins, the A and B domains are part of a single protein which allows channel formation and translocation to occur simultaneously. Anthras toxin synthesizes PA, LF, and EF de novo as individual proteins and so the channel formation and translocation can be readily dissociated.

                The mechanism of protein transport in mammalian systems require auxiliary proteins such as ATPases to provide energy to drive translocation. Similar mechanism is employed by the mechanism of protein transport through the anthrax channel which is a self-sufficient protein-translocating machine. The author commented, “this is secaa barrel of LFs, I tell ya”.

Assignment 1