Replacement of C-Terminal Histidines Uncouples Membrane Insertion and Translocation in Diphtheria Toxin T-Domain

Rodnin, Mykola V.; Kyrychenko, Alexander; Kienker, Paul K.; Sharma, Onkar; Vargas-Uribe, Mauricio; Collier, R. John; Finkelstein, Alan; Ladokhin, Alexey S.

doi:10.1016/j.bpj.2011.10.018

Cited by 29 publications

(105 citation statements)

References 18 publications

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“…Bacterial pore-forming toxins, such as diphtheria, botulinum, and anthrax toxins, are water-soluble at neutral pH, yet they are capable of inserting into lipid bilayers to form ion-permeable channels upon exposure to the acidic pH characteristic of eukaryotic endosomes (29)(30)(31)(32)(33). In this paper, we show that full-length, recombinant APOL1 induces a minor conductance in planar lipid bilayers at acidic pH that can be reversibly amplified by up to 3,000-fold by raising the cis pH (Figs.…”

Section: Discussionmentioning

confidence: 99%

Human trypanolytic factor APOL1 forms pH-gated cation-selective channels in planar lipid bilayers: Relevance to trypanosome lysis

Thomson

Finkelstein

2015

Proc. Natl. Acad. Sci. U.S.A.

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104

158

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Apolipoprotein L-1 (APOL1), the trypanolytic factor of human serum, can lyse several African trypanosome species including Trypanosoma brucei brucei, but not the human-infective pathogens T. brucei rhodesiense and T. brucei gambiense, which are resistant to lysis by human serum. Lysis follows the uptake of APOL1 into acidic endosomes and is apparently caused by colloid-osmotic swelling due to an increased ion permeability of the plasma membrane. Here we demonstrate that nanogram quantities of full-length recombinant APOL1 induce ideally cation-selective macroscopic conductances in planar lipid bilayers. The conductances were highly sensitive to pH: their induction required acidic pH (pH 5.3), but their magnitude could be increased 3,000-fold upon alkalinization of the milieu (pK a = 7.1). We show that this phenomenon can be attributed to the association of APOL1 with the bilayer at acidic pH, followed by the opening of APOL1-induced cationselective channels upon pH neutralization. Furthermore, the conductance increase at neutral pH (but not membrane association at acidic pH) was prevented by the interaction of APOL1 with the serum resistance-associated protein, which is produced by T. brucei rhodesiense and prevents trypanosome lysis by APOL1. These data are consistent with a model of lysis that involves endocytic recycling of APOL1 and the formation of cation-selective channels, at neutral pH, in the parasite plasma membrane.APOL1 | apolipoprotein L-I | pH-gated channel | SRA | African trypanosome

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Section: Discussionmentioning

confidence: 99%

Human trypanolytic factor APOL1 forms pH-gated cation-selective channels in planar lipid bilayers: Relevance to trypanosome lysis

Thomson

Finkelstein

2015

Proc. Natl. Acad. Sci. U.S.A.

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104

158

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“…H257 causes the greatest destabilization of the native structure [113,114]. H322 found between the C-terminal helices TH8 and TH9 plays a role in channel formation and translocation of the N-terminal helices through the lipid bilayer [115,116]. The T domain in the molten globule state has a high affinity (K d < 10 −11 M) for negatively charged phospholipid membranes [104].…”

Section: Membrane Interaction Of the T Domainmentioning

confidence: 98%

Diphtheria toxin

Gillet

Barbier

2015

The Comprehensive Sourcebook of Bacterial Protein Toxins

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“…In contrast, Pathway 1 indicates that the OCS is formed after the translocation, and passageway through the bilayer is formed via an unknown, possibly transient conformation. In order to distinguish between the two pathways, we will use new and published data to compare how various mutations affect the following measures of T-domain activity: (a) conductance in planar bilayers (i.e., formation of the OCS) [37,38,[45][46][47][48], (b) N-terminus translocation in vesicles [37,49], and ultimately (c) cell death assay based on inhibition of protein synthesis in vivo [37,46,48,50]. The starting structure on top corresponds to the crystal structure of the toxin at neutral pH [42], and consists of the C-domain (red), T-domain (helices color-coded according to OCS topology), and R-domain (green).…”

Section: Comparing the Two Translocation Pathwaysmentioning

confidence: 99%

“…[37,46,48]. The data for H322Q, H323Q, and H372Q mutants (color-coded circles) are from Figure 4b and reference [38].…”

Section: Ocs: Critical Intermediate or Byproduct Of Translocationmentioning

confidence: 99%

“…As a result of these studies (Reviewed in [28]), we now understand that the insertion/refolding process occurs along a complex pathway, which includes multiple intermediates with staggered pH-dependent transitions [29][30][31][32][33]. A number of critical titratable residues involved in acid-induced conformational switching have been identified in a series of spectroscopic studies in vitro (e.g., H223 and H257 [32][33][34][35], E349 and D352 [36], and the C-terminal histidines H322, H323, and H372 [35,37,38]). Two recent studies of T-domain conductivity in planar lipid bilayers provided better understanding of the topological arrangements of the T-domain at the late stages of the pathway, at the so-called Open-Channel State (OCS) [39,40].…”

Section: Introductionmentioning

confidence: 99%

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Cellular Entry of Diphtheria Toxin Does Not Require Formation of the Open-Channel State by its Translocation Domain

Rodnin¹,

Vargas-Uribe²,

Ladokhin³

2018

Biophysical Journal

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Cellular entry of diphtheria toxin is a multistage process involving receptor targeting, endocytosis, and translocation of the catalytic domain across the endosomal membrane into the cytosol. The latter is ensured by the translocation (T) domain of the toxin, capable of undergoing conformational refolding and membrane insertion in response to the acidification of the endosomal environment. While numerous now classical studies have demonstrated the formation of an ion-conducting conformation-the Open-Channel State (OCS)-as the final step of the refolding pathway, it remains unclear whether this channel constitutes an in vivo translocation pathway or is a byproduct of the translocation. To address this question, we measure functional activity of known OCS-blocking mutants with H-to-Q replacements of C-terminal histidines of the T-domain. We also test the ability of these mutants to translocate their own N-terminus across lipid bilayers of model vesicles. The results of both experiments indicate that translocation activity does not correlate with previously published OCS activity. Finally, we determined the topology of TH5 helix in membrane-inserted T-domain using W281 fluorescence and its depth-dependent quenching by brominated lipids. Our results indicate that while TH5 becomes a transbilayer helix in a wild-type protein, it fails to insert in the case of the OCS-blocking mutant H322Q. We conclude that the formation of the OCS is not necessary for the functional translocation by the T-domain, at least in the histidine-replacement mutants, suggesting that the OCS is unlikely to constitute a translocation pathway for the cellular entry of diphtheria toxin in vivo.

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Replacement of C-Terminal Histidines Uncouples Membrane Insertion and Translocation in Diphtheria Toxin T-Domain

Cited by 29 publications

References 18 publications

Human trypanolytic factor APOL1 forms pH-gated cation-selective channels in planar lipid bilayers: Relevance to trypanosome lysis

Human trypanolytic factor APOL1 forms pH-gated cation-selective channels in planar lipid bilayers: Relevance to trypanosome lysis

Diphtheria toxin

Cellular Entry of Diphtheria Toxin Does Not Require Formation of the Open-Channel State by its Translocation Domain

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