Limits...
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.

Show MeSH

Related in: MedlinePlus

The primary sequence of LFN (Bragg and Robertson, 1989) with its N-terminal Novagen His6-tag. The orange arrows mark the cysteine mutants investigated in this paper. Acidic residues (aspartates and glutamates) are colored in red, and basic residues (lysines, arginines, and histidines) are in blue. Above the sequence, the red sine waves designate α-helices, the yellow arrows are β-structure, the black lines mark neither α-helices nor β-structure, as determined from the crystal structure of LF (PDB entry 1JKY), and the gray line designates the unresolved unstructured region, residues 1–28.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig1: The primary sequence of LFN (Bragg and Robertson, 1989) with its N-terminal Novagen His6-tag. The orange arrows mark the cysteine mutants investigated in this paper. Acidic residues (aspartates and glutamates) are colored in red, and basic residues (lysines, arginines, and histidines) are in blue. Above the sequence, the red sine waves designate α-helices, the yellow arrows are β-structure, the black lines mark neither α-helices nor β-structure, as determined from the crystal structure of LF (PDB entry 1JKY), and the gray line designates the unresolved unstructured region, residues 1–28.

Mentions: Wild-type (WT) PA (83 kD), its F427A mutant, LFN (residues 1–263 of LF, containing the N-terminal Novagen His6-tag), and its cysteine mutants (Fig. 1) were expressed recombinantly and purified as described previously (Krantz et al., 2004, 2005; Pimental et al., 2004; Zhang et al., 2004b). The heptameric prepore form of PA63 was prepared by nicking PA83 with trypsin and purifying the PA63 heptamer from the smaller 20-kD fragment using anion-exchange chromatography (Cunningham et al., 2002). The MTS-labeling reaction of LFN cysteine mutants was performed by incubation of 100 µl LFN (2 mg/ml) with 3 µl of MTS reagents (20 µg/µl; Toronto Research Chemicals) for 30 min at room temperature in degassed 150 mM NaCl and 20 mM NaPO4, pH 7.2. The reaction was stopped by dialyzing out the MTS reagents at 4°C. To confirm that virtually all of the protein was labeled, MTS-reacted and unreacted protein were incubated with 600 µM maleimide 5,000 polyethylene-glycol (PEG; Sigma-Aldrich) for 30 min at 30°C and then run on an SDS non-reducing acrylamide gel. The unreacted protein suffers a shift on the gel, whereas the MTS-reacted LFN is unaffected (Fig. 2); WT LFN, which has no native cysteines, is also unaffected (not depicted).


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 primary sequence of LFN (Bragg and Robertson, 1989) with its N-terminal Novagen His6-tag. The orange arrows mark the cysteine mutants investigated in this paper. Acidic residues (aspartates and glutamates) are colored in red, and basic residues (lysines, arginines, and histidines) are in blue. Above the sequence, the red sine waves designate α-helices, the yellow arrows are β-structure, the black lines mark neither α-helices nor β-structure, as determined from the crystal structure of LF (PDB entry 1JKY), and the gray line designates the unresolved unstructured region, residues 1–28.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig1: The primary sequence of LFN (Bragg and Robertson, 1989) with its N-terminal Novagen His6-tag. The orange arrows mark the cysteine mutants investigated in this paper. Acidic residues (aspartates and glutamates) are colored in red, and basic residues (lysines, arginines, and histidines) are in blue. Above the sequence, the red sine waves designate α-helices, the yellow arrows are β-structure, the black lines mark neither α-helices nor β-structure, as determined from the crystal structure of LF (PDB entry 1JKY), and the gray line designates the unresolved unstructured region, residues 1–28.
Mentions: Wild-type (WT) PA (83 kD), its F427A mutant, LFN (residues 1–263 of LF, containing the N-terminal Novagen His6-tag), and its cysteine mutants (Fig. 1) were expressed recombinantly and purified as described previously (Krantz et al., 2004, 2005; Pimental et al., 2004; Zhang et al., 2004b). The heptameric prepore form of PA63 was prepared by nicking PA83 with trypsin and purifying the PA63 heptamer from the smaller 20-kD fragment using anion-exchange chromatography (Cunningham et al., 2002). The MTS-labeling reaction of LFN cysteine mutants was performed by incubation of 100 µl LFN (2 mg/ml) with 3 µl of MTS reagents (20 µg/µl; Toronto Research Chemicals) for 30 min at room temperature in degassed 150 mM NaCl and 20 mM NaPO4, pH 7.2. The reaction was stopped by dialyzing out the MTS reagents at 4°C. To confirm that virtually all of the protein was labeled, MTS-reacted and unreacted protein were incubated with 600 µM maleimide 5,000 polyethylene-glycol (PEG; Sigma-Aldrich) for 30 min at 30°C and then run on an SDS non-reducing acrylamide gel. The unreacted protein suffers a shift on the gel, whereas the MTS-reacted LFN is unaffected (Fig. 2); WT LFN, which has no native cysteines, is also unaffected (not depicted).

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