Identification of residues lining the anthrax protective antigen channel

Ericka L. Benson, Paul D. Huynh, Alan Finkelstein, R. John Collier

Research output: Contribution to journalArticle

156 Citations (Scopus)

Abstract

In its activated 63 kDa form, the protective antigen (PA) component of anthrax toxin forms a heptameric prepore, which converts to a pore (channel) in endosomal membranes at low pH and mediates translocation of the toxin's enzymic moieties to the cytosol. It has been proposed that the prepore-to- pore conversion involves a conformational rearrangement of a disordered amphipathic loop (D2L2; residues 302-325), in which loops from the 7 protomers combine to form a transmembrane 14-stranded β barrel. To test this model, we generated Cys substitutions in 24 consecutive residues of the D2L2 loop, formed channels in artificial bilayers with each mutant, and examined changes in channel conductance after adding the thiol-reactive, bilayer- impermeant reagent methanethiosulfonate ethyltrimethylammonium (MTS-ET) to the trans compartment. The rationale for these experiments is that reaction of MTS-ET with a Cys residue adds a positively charged group and therefore would likely reduce channel conductance if the residue were in the ion- conducting pathway. We found alternating reduction and absence of reduction of conductance in consecutive residues over two stretches (residues 302-311 and 316-325). This pattern is consistent with alternating polar and apolar residues of the two stretches projecting into the pore lumen and into the bilayer, respectively. Residues connecting these two stretches (residues 312- 315) were responsive to MTS-ET, consistent with their being in a turn region. Single channels formed by selected mutants (H304C and N306C) showed multiple conductance step changes in response to MTS-ET, consistent with an oligomeric pore. We also found that the binding site for the channel-blocking tetraalkylammonium ions is located cis relative to the inserted D2L2 loops. These findings constitute strong evidence in favor of the model of conversion of the prepore to a 14-stranded β barrel pore and solidify the foundation for studies to understand the mechanism of translocation by anthrax toxin.

Original languageEnglish (US)
Pages (from-to)3941-3948
Number of pages8
JournalBiochemistry
Volume37
Issue number11
DOIs
StatePublished - Mar 17 1998

Fingerprint

Linings
Ions
Protein Subunits
Sulfhydryl Compounds
Cytosol
Substitution reactions
Binding Sites
Membranes
Antigens
anthrax toxin
methanethiosulfonate
trimethylethylammonium
Experiments

ASJC Scopus subject areas

  • Biochemistry

Cite this

Benson, E. L., Huynh, P. D., Finkelstein, A., & Collier, R. J. (1998). Identification of residues lining the anthrax protective antigen channel. Biochemistry, 37(11), 3941-3948. https://doi.org/10.1021/bi972657b

Identification of residues lining the anthrax protective antigen channel. / Benson, Ericka L.; Huynh, Paul D.; Finkelstein, Alan; Collier, R. John.

In: Biochemistry, Vol. 37, No. 11, 17.03.1998, p. 3941-3948.

Research output: Contribution to journalArticle

Benson, EL, Huynh, PD, Finkelstein, A & Collier, RJ 1998, 'Identification of residues lining the anthrax protective antigen channel', Biochemistry, vol. 37, no. 11, pp. 3941-3948. https://doi.org/10.1021/bi972657b
Benson EL, Huynh PD, Finkelstein A, Collier RJ. Identification of residues lining the anthrax protective antigen channel. Biochemistry. 1998 Mar 17;37(11):3941-3948. https://doi.org/10.1021/bi972657b
Benson, Ericka L. ; Huynh, Paul D. ; Finkelstein, Alan ; Collier, R. John. / Identification of residues lining the anthrax protective antigen channel. In: Biochemistry. 1998 ; Vol. 37, No. 11. pp. 3941-3948.
@article{70f91a8ee94c471eb6df92499da085d5,
title = "Identification of residues lining the anthrax protective antigen channel",
abstract = "In its activated 63 kDa form, the protective antigen (PA) component of anthrax toxin forms a heptameric prepore, which converts to a pore (channel) in endosomal membranes at low pH and mediates translocation of the toxin's enzymic moieties to the cytosol. It has been proposed that the prepore-to- pore conversion involves a conformational rearrangement of a disordered amphipathic loop (D2L2; residues 302-325), in which loops from the 7 protomers combine to form a transmembrane 14-stranded β barrel. To test this model, we generated Cys substitutions in 24 consecutive residues of the D2L2 loop, formed channels in artificial bilayers with each mutant, and examined changes in channel conductance after adding the thiol-reactive, bilayer- impermeant reagent methanethiosulfonate ethyltrimethylammonium (MTS-ET) to the trans compartment. The rationale for these experiments is that reaction of MTS-ET with a Cys residue adds a positively charged group and therefore would likely reduce channel conductance if the residue were in the ion- conducting pathway. We found alternating reduction and absence of reduction of conductance in consecutive residues over two stretches (residues 302-311 and 316-325). This pattern is consistent with alternating polar and apolar residues of the two stretches projecting into the pore lumen and into the bilayer, respectively. Residues connecting these two stretches (residues 312- 315) were responsive to MTS-ET, consistent with their being in a turn region. Single channels formed by selected mutants (H304C and N306C) showed multiple conductance step changes in response to MTS-ET, consistent with an oligomeric pore. We also found that the binding site for the channel-blocking tetraalkylammonium ions is located cis relative to the inserted D2L2 loops. These findings constitute strong evidence in favor of the model of conversion of the prepore to a 14-stranded β barrel pore and solidify the foundation for studies to understand the mechanism of translocation by anthrax toxin.",
author = "Benson, {Ericka L.} and Huynh, {Paul D.} and Alan Finkelstein and Collier, {R. John}",
year = "1998",
month = "3",
day = "17",
doi = "10.1021/bi972657b",
language = "English (US)",
volume = "37",
pages = "3941--3948",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "11",

}

TY - JOUR

T1 - Identification of residues lining the anthrax protective antigen channel

AU - Benson, Ericka L.

AU - Huynh, Paul D.

AU - Finkelstein, Alan

AU - Collier, R. John

PY - 1998/3/17

Y1 - 1998/3/17

N2 - In its activated 63 kDa form, the protective antigen (PA) component of anthrax toxin forms a heptameric prepore, which converts to a pore (channel) in endosomal membranes at low pH and mediates translocation of the toxin's enzymic moieties to the cytosol. It has been proposed that the prepore-to- pore conversion involves a conformational rearrangement of a disordered amphipathic loop (D2L2; residues 302-325), in which loops from the 7 protomers combine to form a transmembrane 14-stranded β barrel. To test this model, we generated Cys substitutions in 24 consecutive residues of the D2L2 loop, formed channels in artificial bilayers with each mutant, and examined changes in channel conductance after adding the thiol-reactive, bilayer- impermeant reagent methanethiosulfonate ethyltrimethylammonium (MTS-ET) to the trans compartment. The rationale for these experiments is that reaction of MTS-ET with a Cys residue adds a positively charged group and therefore would likely reduce channel conductance if the residue were in the ion- conducting pathway. We found alternating reduction and absence of reduction of conductance in consecutive residues over two stretches (residues 302-311 and 316-325). This pattern is consistent with alternating polar and apolar residues of the two stretches projecting into the pore lumen and into the bilayer, respectively. Residues connecting these two stretches (residues 312- 315) were responsive to MTS-ET, consistent with their being in a turn region. Single channels formed by selected mutants (H304C and N306C) showed multiple conductance step changes in response to MTS-ET, consistent with an oligomeric pore. We also found that the binding site for the channel-blocking tetraalkylammonium ions is located cis relative to the inserted D2L2 loops. These findings constitute strong evidence in favor of the model of conversion of the prepore to a 14-stranded β barrel pore and solidify the foundation for studies to understand the mechanism of translocation by anthrax toxin.

AB - In its activated 63 kDa form, the protective antigen (PA) component of anthrax toxin forms a heptameric prepore, which converts to a pore (channel) in endosomal membranes at low pH and mediates translocation of the toxin's enzymic moieties to the cytosol. It has been proposed that the prepore-to- pore conversion involves a conformational rearrangement of a disordered amphipathic loop (D2L2; residues 302-325), in which loops from the 7 protomers combine to form a transmembrane 14-stranded β barrel. To test this model, we generated Cys substitutions in 24 consecutive residues of the D2L2 loop, formed channels in artificial bilayers with each mutant, and examined changes in channel conductance after adding the thiol-reactive, bilayer- impermeant reagent methanethiosulfonate ethyltrimethylammonium (MTS-ET) to the trans compartment. The rationale for these experiments is that reaction of MTS-ET with a Cys residue adds a positively charged group and therefore would likely reduce channel conductance if the residue were in the ion- conducting pathway. We found alternating reduction and absence of reduction of conductance in consecutive residues over two stretches (residues 302-311 and 316-325). This pattern is consistent with alternating polar and apolar residues of the two stretches projecting into the pore lumen and into the bilayer, respectively. Residues connecting these two stretches (residues 312- 315) were responsive to MTS-ET, consistent with their being in a turn region. Single channels formed by selected mutants (H304C and N306C) showed multiple conductance step changes in response to MTS-ET, consistent with an oligomeric pore. We also found that the binding site for the channel-blocking tetraalkylammonium ions is located cis relative to the inserted D2L2 loops. These findings constitute strong evidence in favor of the model of conversion of the prepore to a 14-stranded β barrel pore and solidify the foundation for studies to understand the mechanism of translocation by anthrax toxin.

UR - http://www.scopus.com/inward/record.url?scp=0032539990&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0032539990&partnerID=8YFLogxK

U2 - 10.1021/bi972657b

DO - 10.1021/bi972657b

M3 - Article

VL - 37

SP - 3941

EP - 3948

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 11

ER -