scholarly journals Structure of the Diphtheria Toxin at Acidic pH: Implications for the Conformational Switching of the Translocation Domain

Toxins ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 704
Author(s):  
Mykola V. Rodnin ◽  
Maithri M. Kashipathy ◽  
Alexander Kyrychenko ◽  
Kevin P. Battaile ◽  
Scott Lovell ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Diphtheria Toxin ◽  
Catalytic Domain ◽  
Conformational Transition ◽  
Published Data ◽  
Acidic Ph ◽  
X Ray Crystallography ◽  
Binding Domains ◽  
Endosomal Acidification ◽  
T Domain

Diphtheria toxin, an exotoxin secreted by Corynebacterium that causes disease in humans by inhibiting protein synthesis, enters the cell via receptor-mediated endocytosis. The subsequent endosomal acidification triggers a series of conformational changes, resulting in the refolding and membrane insertion of the translocation (T-)domain and ultimately leading to the translocation of the catalytic domain into the cytoplasm. Here, we use X-ray crystallography along with circular dichroism and fluorescence spectroscopy to gain insight into the mechanism of the early stages of pH-dependent conformational transition. For the first time, we present the high-resolution structure of the diphtheria toxin at a mildly acidic pH (5–6) and compare it to the structure at neutral pH (7). We demonstrate that neither catalytic nor receptor-binding domains change their structure upon this acidification, while the T-domain undergoes a conformational change that results in the unfolding of the TH2–3 helices. Surprisingly, the TH1 helix maintains its conformation in the crystal of the full-length toxin even at pH 5. This contrasts with the evidence from the new and previously published data, obtained by spectroscopic measurements and molecular dynamics computer simulations, which indicate the refolding of TH1 upon the acidification of the isolated T-domain. The overall results imply that the membrane interactions of the T-domain are critical in ensuring the proper conformational changes required for the preparation of the diphtheria toxin for the cellular entry.

scholarly journals A structural framework for unidirectional transport by a bacterial ABC exporter

2020 ◽  
Vol 117 (32) ◽  
pp. 19228-19236
Author(s):  
Chengcheng Fan ◽  
Jens T. Kaiser ◽  
Douglas C. Rees
Keyword(s):  
Conformational Changes ◽  
Iron Homeostasis ◽  
Atp Hydrolysis ◽  
Transmembrane Helix ◽  
Reverse Direction ◽  
Conformational States ◽  
X Ray Crystallography ◽  
Binding Domains ◽  
Structural Framework ◽  
Glutathione Derivatives

The ATP-binding cassette (ABC) transporter of mitochondria (Atm1) mediates iron homeostasis in eukaryotes, while the prokaryotic homolog fromNovosphingobium aromaticivorans(NaAtm1) can export glutathione derivatives and confer protection against heavy-metal toxicity. To establish the structural framework underlying theNaAtm1 transport mechanism, we determined eight structures by X-ray crystallography and single-particle cryo-electron microscopy in distinct conformational states, stabilized by individual disulfide crosslinks and nucleotides. AsNaAtm1 progresses through the transport cycle, conformational changes in transmembrane helix 6 (TM6) alter the glutathione-binding site and the associated substrate-binding cavity. Significantly, kinking of TM6 in the post-ATP hydrolysis state stabilized by MgADPVO4eliminates this cavity, precluding uptake of glutathione derivatives. The presence of this cavity during the transition from the inward-facing to outward-facing conformational states, and its absence in the reverse direction, thereby provide an elegant and conceptually simple mechanism for enforcing the export directionality of transport byNaAtm1. One of the disulfide crosslinkedNaAtm1 variants characterized in this work retains significant glutathione transport activity, suggesting that ATP hydrolysis and substrate transport by Atm1 may involve a limited set of conformational states with minimal separation of the nucleotide-binding domains in the inward-facing conformation.

scholarly journals Topography of Diphtheria Toxin's T Domain in the Open Channel State

2000 ◽  
Vol 115 (4) ◽  
pp. 421-434 ◽  
Author(s):  
Lisa Senzel ◽  
Michael Gordon ◽  
Robert O. Blaustein ◽  
K. Joon Oh ◽  
R. John Collier ◽  
...  
Keyword(s):  
Diphtheria Toxin ◽  
Open Channel ◽  
Catalytic Domain ◽  
Water Soluble ◽  
Soluble Form ◽  
Crystallographic Structure ◽  
Channel State ◽  
Amino Terminal ◽  
T Domain ◽  
Membrane Spanning

When diphtheria toxin encounters a low pH environment, the channel-forming T domain undergoes a poorly understood conformational change that allows for both its own membrane insertion and the translocation of the toxin's catalytic domain across the membrane. From the crystallographic structure of the water-soluble form of diphtheria toxin, a “double dagger” model was proposed in which two transmembrane helical hairpins, TH5-7 and TH8-9, anchor the T domain in the membrane. In this paper, we report the topography of the T domain in the open channel state. This topography was derived from experiments in which either a hexahistidine (H6) tag or biotin moiety was attached at residues that were mutated to cysteines. From the sign of the voltage gating induced by the H6 tag and the accessibility of the biotinylated residues to streptavidin added to the cis or trans side of the membrane, we determined which segments of the T domain are on the cis or trans side of the membrane and, consequently, which segments span the membrane. We find that there are three membrane-spanning segments. Two of them are in the channel-forming piece of the T domain, near its carboxy terminal end, and correspond to one of the proposed “daggers,” TH8-9. The other membrane-spanning segment roughly corresponds to only TH5 of the TH5-7 dagger, with the rest of that region lying on or near the cis surface. We also find that, in association with channel formation, the amino terminal third of the T domain, a hydrophilic stretch of ∼70 residues, is translocated across the membrane to the trans side.

scholarly journals The Number of Subunits Comprising the Channel Formed by the T Domain of Diphtheria Toxin

2001 ◽  
Vol 118 (5) ◽  
pp. 471-480 ◽  
Author(s):  
Michael Gordon ◽  
Alan Finkelstein
Keyword(s):  
Lipid Bilayers ◽  
Diphtheria Toxin ◽  
Catalytic Domain ◽  
Low Ph ◽  
Voltage Gating ◽  
Single Channels ◽  
Planar Lipid Bilayers ◽  
Transmembrane Segments ◽  
T Domain ◽  
Single Subunit

In the presence of a low pH environment, the channel-forming T domain of diphtheria toxin undergoes a conformational change that allows for both its own insertion into planar lipid bilayers and the translocation of the toxin's catalytic domain across them. Given that the T domain contributes only three transmembrane segments, and the channel is permeable to ions as large as glucosamine+ and NAD−, it would appear that the channel must be a multimer. Yet, there is substantial circumstantial evidence that the channel may be formed from a single subunit. To test the hypothesis that the channel formed by the T domain of diphtheria toxin is monomeric, we made mixtures of two T domain constructs whose voltage-gating characteristics differ, and then observed the gating behavior of the mixture's single channels in planar lipid bilayers. One of these constructs contained an NH2-terminal hexahistidine (H6) tag that blocks the channel at negative voltages; the other contained a COOH-terminal H6 tag that blocks the channel at positive voltages. If the channel is constructed from multiple T domain subunits, one expects to see a population of single channels from this mixture that are blocked at both positive and negative voltages. The observed single channels were blocked at either negative or positive voltages, but never both. Therefore, we conclude that the T domain channel is monomeric.

scholarly journals Mapping the membrane topography of the TH6–TH7 segment of the diphtheria toxin T-domain channel

2015 ◽  
Vol 145 (2) ◽  
pp. 107-125 ◽  
Author(s):  
Paul K. Kienker ◽  
Zhengyan Wu ◽  
Alan Finkelstein
Keyword(s):  
Lipid Bilayers ◽  
Diphtheria Toxin ◽  
Catalytic Domain ◽  
Single Channel ◽  
Reaction Rates ◽  
Cysteine Residue ◽  
Channel Conductance ◽  
Amino Terminal ◽  
Substituted Cysteine Accessibility ◽  
T Domain

Low pH triggers the translocation domain of diphtheria toxin (T-domain), which contains 10 α helices, to insert into a planar lipid bilayer membrane, form a transmembrane channel, and translocate the attached catalytic domain across the membrane. Three T-domain helices, corresponding to TH5, TH8, and TH9 in the aqueous crystal structure, form transmembrane segments in the open-channel state; the amino-terminal region, TH1–TH4, translocates across the membrane to the trans side. Residues near either end of the TH6–TH7 segment are not translocated, remaining on the cis side of the membrane; because the intervening 25-residue sequence is too short to form a transmembrane α-helical hairpin, it was concluded that the TH6–TH7 segment resides at the cis interface. Now we have examined this segment further, using the substituted-cysteine accessibility method. We constructed a series of 18 mutant T-domains with single cysteine residues at positions in TH6–TH7, monitored their channel formation in planar lipid bilayers, and probed for an effect of thiol-specific reagents on the channel conductance. For 10 of the mutants, the reagent caused a change in the single-channel conductance, indicating that the introduced cysteine residue was exposed within the channel lumen. For several of these mutants, we verified that the reactions occurred primarily in the open state, rather than in the flicker-closed state. We also established that blocking of the channel by an amino-terminal hexahistidine tag could protect mutants from reaction. Finally, we compared the reaction rates of reagent added to the cis and trans sides to quantify the residue’s accessibility from either side. This analysis revealed abrupt changes in cis- versus trans-side accessibility, suggesting that the TH6–TH7 segment forms a constriction that occupies a small portion of the total channel length. We also determined that this constriction is located near the middle of the TH8 helix.

scholarly journals Three-color single-molecule imaging reveals conformational dynamics of dynein undergoing motility

2020 ◽  
Author(s):  
Stefan Niekamp ◽  
Nico Stuurman ◽  
Nan Zhang ◽  
Ronald D. Vale
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
In Silico ◽  
Conformational Transition ◽  
Conformational Dynamics ◽  
Motor Protein ◽  
Single Molecule Imaging ◽  
Microtubule Binding ◽  
Binding Domains ◽  
Single Molecule Studies

The motor protein dynein undergoes coordinated conformational changes of its domains during motility along microtubules. Previous single-molecule studies analyzed the motion of the AAA rings of the dynein homodimer, but not the distal microtubule binding domains (MTBD) that step along the track. Here, we simultaneously tracked two MTBDs and one AAA ring of a single dynein, as it undergoes hundreds of steps with nanometer precision using three-color imaging. We show that the AAA ring and the MTBDs do not always step simultaneously and can take different sized steps. This variability in the movement between AAA ring and MTBD results in an unexpectedly large number of conformational states of dynein during motility. Extracting data on conformational transition biases, we could accurately model dynein stepping in silico. Our results reveal that the flexibility between major dynein domains is critical for dynein motility.

scholarly journals Three-color single-molecule imaging reveals conformational dynamics of dynein undergoing motility

2021 ◽  
Vol 118 (31) ◽  
pp. e2101391118
Author(s):  
Stefan Niekamp ◽  
Nico Stuurman ◽  
Nan Zhang ◽  
Ronald D. Vale
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
In Silico ◽  
Conformational Transition ◽  
Conformational Dynamics ◽  
Motor Protein ◽  
Single Molecule Imaging ◽  
Microtubule Binding ◽  
Binding Domains ◽  
Single Molecule Studies

The motor protein dynein undergoes coordinated conformational changes of its domains during motility along microtubules. Previous single-molecule studies analyzed the motion of the AAA rings of the dynein homodimer, but not the distal microtubule-binding domains (MTBDs) that step along the track. Here, we simultaneously tracked with nanometer precision two MTBDs and one AAA ring of a single dynein as it underwent hundreds of steps using three-color imaging. We show that the AAA ring and the MTBDs do not always step simultaneously and can take differently sized steps. This variability in the movement between the AAA ring and MTBDs results in an unexpectedly large number of conformational states of dynein during motility. Extracting data on conformational transition biases, we could accurately model dynein stepping in silico. Our results reveal that the flexibility between major dynein domains is critical for dynein motility.

scholarly journals pH-Induced Conformational Changes inClostridium difficile Toxin B

2000 ◽  
Vol 68 (5) ◽  
pp. 2470-2474 ◽  
Author(s):  
Maen Qa'Dan ◽  
Lea M. Spyres ◽  
Jimmy D. Ballard
Keyword(s):  
Conformational Changes ◽  
Mammalian Cells ◽  
Structural Changes ◽  
Tryptophan Fluorescence ◽  
Acidic Ph ◽  
Bafilomycin A1 ◽  
Toxin B ◽  
Endosomal Acidification ◽  
Specific Protease ◽  
The Impact

ABSTRACT Toxin B from Clostridium difficile is a monoglucosylating toxin that targets substrates within the cytosol of mammalian cells. In this study, we investigated the impact of acidic pH on cytosolic entry and structural changes within toxin B. Bafilomycin A1 was used to block endosomal acidification and subsequent toxin B translocation. Cytopathic effects could be completely blocked by addition of bafilomycin A1 up to 20 min following toxin treatment. Furthermore, providing a low extracellular pH could circumvent the effect of bafilomycin A1 and other lysosomotropic agents. Acid pH-induced structural changes were monitored by using the fluorescent probe 2-(p-toluidinyl) naphthalene-6-sulfonic acid, sodium salt (TNS), inherent tryptophan fluorescence, and relative susceptibility to a specific protease. As the toxin was exposed to lower pH there was an increase in TNS fluorescence, suggesting the exposure of hydrophobic domains by toxin B. The change in hydrophobicity appeared to be reversible, since returning the pH to neutrality abrogated TNS fluorescence. Furthermore, tryptophan fluorescence was quenched at the acidic pH, indicating that domains may have been moving into more aqueous environments. Toxin B also demonstrated variable susceptibility to Staphylococcus aureus V8 protease at neutral and acidic pH, further suggesting pH-induced structural changes in this protein.

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