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Evidence for a proton-protein symport mechanism in the anthrax toxin channel.

Basilio D, Juris SJ, Collier RJ, Finkelstein A - J. Gen. Physiol. (2009)

Bottom Line: Therefore, the translocated species is positively charged.Here, we provide further evidence of such a mechanism by showing that if only one SO(3)(-), which is essentially not titratable, is introduced at most positions in LF(N), through the reaction of an introduced cysteine residue at those positions with 2-sulfonato-ethyl-methanethiosulfonate, voltage-driven LF(N) translocation is drastically inhibited.We also find that a site that disfavors the entry of negatively charged residues into the (PA(63))(7) channel resides at or near its Phi-clamp, the ring of seven phenylalanines near the channel's entrance.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY 10461, USA. dbseyler@aecom.yu.edu

ABSTRACT
The toxin produced by Bacillus anthracis, the causative agent of anthrax, is composed of three proteins: a translocase heptameric channel, (PA(63))(7), formed from protective antigen (PA), which allows the other two proteins, lethal and edema factors (LF and EF), to translocate across a host cell's endosomal membrane, disrupting cellular homeostasis. It has been shown that (PA(63))(7) incorporated into planar phospholipid bilayer membranes forms a channel capable of transporting LF and EF. Protein translocation through the channel is driven by a proton electrochemical potential gradient on a time scale of seconds. A paradoxical aspect of this is that although LF(N) (the N-terminal 263 residues of LF), on which most of our experiments were performed, has a net negative charge, it is driven through the channel by a cis-positive voltage. We have explained this by claiming that the (PA(63))(7) channel strongly disfavors the entry of negatively charged residues on proteins to be translocated, and hence the aspartates and glutamates on LF(N) enter protonated (i.e., neutralized). Therefore, the translocated species is positively charged. Upon exiting the channel, the protons that were picked up from the cis solution are released into the trans solution, thereby making this a proton-protein symporter. Here, we provide further evidence of such a mechanism by showing that if only one SO(3)(-), which is essentially not titratable, is introduced at most positions in LF(N), through the reaction of an introduced cysteine residue at those positions with 2-sulfonato-ethyl-methanethiosulfonate, voltage-driven LF(N) translocation is drastically inhibited. We also find that a site that disfavors the entry of negatively charged residues into the (PA(63))(7) channel resides at or near its Phi-clamp, the ring of seven phenylalanines near the channel's entrance.

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Related in: MedlinePlus

The effect of introducing one SO3− (by reacting an LFN cysteine mutant with MTS-ES) on the translocation rate of LFN. The panels show the normalized rise of conductance (a reflection of translocation of LFN through the (PA63)7 channel) that occurred when, after the perfusion of the indicated MTS-reacted LFN cysteine mutant out of the cis compartment, the voltage was stepped from +20 to +55 mV. Note the very slow translocation of the MTS-ES–reacted LFN (red). In contrast, the translocation rates of the MTS-ET–reacted (blue) and the MTS-ACE–reacted LFN (black) were essentially the same as that of WT LFN (which is not shown in the panels for clarity), with a half-time of ∼5 s.
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fig3: The effect of introducing one SO3− (by reacting an LFN cysteine mutant with MTS-ES) on the translocation rate of LFN. The panels show the normalized rise of conductance (a reflection of translocation of LFN through the (PA63)7 channel) that occurred when, after the perfusion of the indicated MTS-reacted LFN cysteine mutant out of the cis compartment, the voltage was stepped from +20 to +55 mV. Note the very slow translocation of the MTS-ES–reacted LFN (red). In contrast, the translocation rates of the MTS-ET–reacted (blue) and the MTS-ACE–reacted LFN (black) were essentially the same as that of WT LFN (which is not shown in the panels for clarity), with a half-time of ∼5 s.

Mentions: In Figs. 3–6, what is plotted is the normalized conductance versus time. With the exception of the MTS-ES–reacted LFN cysteine mutants that never reached a constant value, conductances were normalized to the level obtained after unblocking at +55 mV was completed. In general, this conductance level was ≥90% of the conductance level before blocking by LFN. For the MTS-ES–reacted LFN mutants, after the 55 mV was applied for the length of time indicated in the figures, the voltage was stepped for 30 s to −80 mV, and then back to +20 mV. Negative voltages drive LFN out of the channel back to the cis side, so that the conductance immediately obtained at +20 mV (before the slow reblocking of the channels by still-attached LFN) represents that of unblocked channels. Conductances were normalized to this level, which was ≥90% of the conductance level before blocking by LFN.


Evidence for a proton-protein symport mechanism in the anthrax toxin channel.

Basilio D, Juris SJ, Collier RJ, Finkelstein A - J. Gen. Physiol. (2009)

The effect of introducing one SO3− (by reacting an LFN cysteine mutant with MTS-ES) on the translocation rate of LFN. The panels show the normalized rise of conductance (a reflection of translocation of LFN through the (PA63)7 channel) that occurred when, after the perfusion of the indicated MTS-reacted LFN cysteine mutant out of the cis compartment, the voltage was stepped from +20 to +55 mV. Note the very slow translocation of the MTS-ES–reacted LFN (red). In contrast, the translocation rates of the MTS-ET–reacted (blue) and the MTS-ACE–reacted LFN (black) were essentially the same as that of WT LFN (which is not shown in the panels for clarity), with a half-time of ∼5 s.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2654084&req=5

fig3: The effect of introducing one SO3− (by reacting an LFN cysteine mutant with MTS-ES) on the translocation rate of LFN. The panels show the normalized rise of conductance (a reflection of translocation of LFN through the (PA63)7 channel) that occurred when, after the perfusion of the indicated MTS-reacted LFN cysteine mutant out of the cis compartment, the voltage was stepped from +20 to +55 mV. Note the very slow translocation of the MTS-ES–reacted LFN (red). In contrast, the translocation rates of the MTS-ET–reacted (blue) and the MTS-ACE–reacted LFN (black) were essentially the same as that of WT LFN (which is not shown in the panels for clarity), with a half-time of ∼5 s.
Mentions: In Figs. 3–6, what is plotted is the normalized conductance versus time. With the exception of the MTS-ES–reacted LFN cysteine mutants that never reached a constant value, conductances were normalized to the level obtained after unblocking at +55 mV was completed. In general, this conductance level was ≥90% of the conductance level before blocking by LFN. For the MTS-ES–reacted LFN mutants, after the 55 mV was applied for the length of time indicated in the figures, the voltage was stepped for 30 s to −80 mV, and then back to +20 mV. Negative voltages drive LFN out of the channel back to the cis side, so that the conductance immediately obtained at +20 mV (before the slow reblocking of the channels by still-attached LFN) represents that of unblocked channels. Conductances were normalized to this level, which was ≥90% of the conductance level before blocking by LFN.

Bottom Line: Therefore, the translocated species is positively charged.Here, we provide further evidence of such a mechanism by showing that if only one SO(3)(-), which is essentially not titratable, is introduced at most positions in LF(N), through the reaction of an introduced cysteine residue at those positions with 2-sulfonato-ethyl-methanethiosulfonate, voltage-driven LF(N) translocation is drastically inhibited.We also find that a site that disfavors the entry of negatively charged residues into the (PA(63))(7) channel resides at or near its Phi-clamp, the ring of seven phenylalanines near the channel's entrance.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY 10461, USA. dbseyler@aecom.yu.edu

ABSTRACT
The toxin produced by Bacillus anthracis, the causative agent of anthrax, is composed of three proteins: a translocase heptameric channel, (PA(63))(7), formed from protective antigen (PA), which allows the other two proteins, lethal and edema factors (LF and EF), to translocate across a host cell's endosomal membrane, disrupting cellular homeostasis. It has been shown that (PA(63))(7) incorporated into planar phospholipid bilayer membranes forms a channel capable of transporting LF and EF. Protein translocation through the channel is driven by a proton electrochemical potential gradient on a time scale of seconds. A paradoxical aspect of this is that although LF(N) (the N-terminal 263 residues of LF), on which most of our experiments were performed, has a net negative charge, it is driven through the channel by a cis-positive voltage. We have explained this by claiming that the (PA(63))(7) channel strongly disfavors the entry of negatively charged residues on proteins to be translocated, and hence the aspartates and glutamates on LF(N) enter protonated (i.e., neutralized). Therefore, the translocated species is positively charged. Upon exiting the channel, the protons that were picked up from the cis solution are released into the trans solution, thereby making this a proton-protein symporter. Here, we provide further evidence of such a mechanism by showing that if only one SO(3)(-), which is essentially not titratable, is introduced at most positions in LF(N), through the reaction of an introduced cysteine residue at those positions with 2-sulfonato-ethyl-methanethiosulfonate, voltage-driven LF(N) translocation is drastically inhibited. We also find that a site that disfavors the entry of negatively charged residues into the (PA(63))(7) channel resides at or near its Phi-clamp, the ring of seven phenylalanines near the channel's entrance.

Show MeSH
Related in: MedlinePlus