Mauricio Vargas-Uribe

pH-triggered conformational switching of the diphtheria toxin T-domain: the roles of N-terminal histidines.

pH-induced conformational switching is essential for functioning of diphtheria toxin, which undergoes a membrane insertion/translocation transition triggered by endosomal acidification as a key step of cellular entry. In order to establish the sequence of molecular rearrangements and side-chain protonation accompanying the formation of the membrane-competent state of the toxins translocation (T) domain, we have developed and applied an integrated approach that combines multiple techniques of computational chemistry [e.g., long-microsecond-range, all-atom molecular dynamics (MD) simulations; continuum electrostatics calculations; and thermodynamic integration (TI)] with several experimental techniques of fluorescence spectroscopy. TI calculations indicate that protonation of H257 causes the greatest destabilization of the native structure (6.9 kcal/mol), which is consistent with our early mutagenesis results. Extensive equilibrium MD simulations with a combined length of over 8 μs demonstrate that histidine protonation, while not accompanied by the loss of structural compactness of the T-domain, nevertheless results in substantial molecular rearrangements characterized by the partial loss of secondary structure due to unfolding of helices TH1 and TH2 and the loss of close contact between the C- and N-terminal segments. The structural changes accompanying the formation of the membrane-competent state ensure an easier exposure of the internal hydrophobic hairpin formed by helices TH8 and TH9, in preparation for its subsequent transmembrane insertion.

Crucial Role of H322 in the Folding of Diphtheria Toxin T-Domain into the Open-Channel State

The translocation (T) domain plays a key role in the entry of diphtheria toxin into the cell. Upon endosomal acidification, the T-domain undergoes a series of conformational changes that lead to its membrane insertion and formation of a channel. Recently, we have reported that the triple replacement of C-terminal histidines H322, H323, and H372 with glutamines prevents the formation of open channels in planar lipid bilayers. Here, we report that this effect is primarily due to the mutation of H322. We further examine the relationship between the loss of functionality and membrane folding in a series of mutants with C-terminal histidine substitutions using spectroscopic assays. The membrane insertion pathway for the mutants differs from that of the wild type as revealed by the membrane-induced red shift of tryptophan fluorescence at pH 6.0-6.5. T-Domain mutants with replacements at H323 and H372, but not at H322, regain a wild-type-like spectroscopic signature upon further acidification. Circular dichroism measurements confirm that affected mutants misfold during insertion into vesicles. Conductance measurements reveal that substituting H322 dramatically reduces the numbers of properly folded channels in a planar bilayer, but the properties of the active channels appear to be unaltered. We propose that H322 plays an important role in the formation of open channels and is involved in guiding the proper insertion of the N-terminal region of the T-domain into the membrane.

Comparison of Membrane Insertion Pathways of the Apoptotic Regulator Bcl-xL and the Diphtheria Toxin Translocation Domain

The diphtheria toxin translocation domain (T-domain) and the apoptotic repressor Bcl-xL are membrane proteins that adopt their final topology by switching folds from a water-soluble to a membrane-inserted state. While the exact molecular mechanisms of this transition are not clearly understood in either case, the similarity in the structures of soluble states of the T-domain and Bcl-xL led to the suggestion that their membrane insertion pathways will be similar, as well. Previously, we have applied an array of spectroscopic methods to characterize the pH-triggered refolding and membrane insertion of the diphtheria toxin T-domain. Here, we use the same set of methods to describe the membrane insertion pathway of Bcl-xL, which allows us to make a direct comparison between both systems with respect to the thermodynamic stability in solution, pH-dependent membrane association, and transmembrane insertion. Thermal denaturation measured by circular dichroism indicates that, unlike the T-domain, Bcl-xL does not undergo a pH-dependent destabilization of the structure. Förster resonance energy transfer measurements demonstrate that Bcl-xL undergoes reversible membrane association modulated by the presence of anionic lipids, suggesting that formation of the membrane-competent form occurs close to the membrane interface. Membrane insertion of the main hydrophobic helical hairpin of Bcl-xL, α5-α6, was studied by site-selective attachment of environment-sensitive dye NBD. In contrast to the insertion of the corresponding TH8-TH9 hairpin into the T-domain, insertion of α5-α6 was found not to depend strongly on the presence of anionic lipids. Taken together, our results indicate that while Bcl-xL and the T-domain share structural similarities, their modes of conformational switching and membrane insertion pathways are distinctly different.

Role of Acidic Residues in Helices TH8-TH9 in Membrane Interactions of the Diphtheria Toxin T Domain

The pH-triggered membrane insertion of the diphtheria toxin translocation domain (T domain) results in transferring the catalytic domain into the cytosol, which is relevant to potential biomedical applications as a cargo-delivery system. Protonation of residues is suggested to play a key role in the process, and residues E349, D352 and E362 are of particular interest because of their location within the membrane insertion unit TH8–TH9. We have used various spectroscopic, computational and functional assays to characterize the properties of the T domain carrying the double mutation E349Q/D352N or the single mutation E362Q. Vesicle leakage measurements indicate that both mutants interact with the membrane under less acidic conditions than the wild-type. Thermal unfolding and fluorescence measurements, complemented with molecular dynamics simulations, suggest that the mutant E362Q is more susceptible to acid destabilization because of disruption of native intramolecular contacts. Fluorescence experiments show that removal of the charge in E362Q, and not in E349Q/D352N, is important for insertion of TH8–TH9. Both mutants adopt a final functional state upon further acidification. We conclude that these acidic residues are involved in the pH-dependent action of the T domain, and their replacements can be used for fine tuning the pH range of membrane interactions.

Membrane Association of the Diphtheria Toxin Translocation Domain Studied by Coarse-Grained Simulations and Experiment

Diphtheria toxin translocation (T) domain inserts in lipid bilayers upon acidification of the environment. Computational and experimental studies have suggested that low pH triggers a conformational change of the T-domain in solution preceding membrane binding. The refolded membrane-competent state was modeled to be compact and mostly retain globular structure. In the present work, we investigate how this refolded state interacts with membrane interfaces in the early steps of T-domain’s membrane association. Coarse-grained molecular dynamics simulations suggest two distinct membrane-bound conformations of the T-domain in the presence of bilayers composed of a mixture of zwitteronic and anionic phospholipids (POPC:POPG with a 1:3 molar ratio). Both membrane-bound conformations show a common near parallel orientation of hydrophobic helices TH8–TH9 relative to the membrane plane. The most frequently observed membrane-bound conformation is stabilized by electrostatic interactions between the N-terminal segment of the protein and the membrane interface. The second membrane-bound conformation is stabilized by hydrophobic interactions between protein residues and lipid acyl chains, which facilitate deeper protein insertion in the membrane interface. A theoretical estimate of a free energy of binding of a membrane-competent T-domain to the membrane is provided.Graphical Abstract

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

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.

Lipid-Dependent Modulation of Membrane Recruitment and Protein-Protein Interactions in BCL-2 Family of Apoptotic Regulators

Permeabilization of the mitochondrial outer membrane during apoptosis is regulated by interactions between BCL-2 proteins, including tBid, Bax and Bcl-xL. The anti-apoptotic member Bcl-xL exists in at least three states: a water-soluble cytosolic state, a membrane anchored state with a C-terminal helix acting as the anchor, and a membrane inserted state. Different factors have been suggested to modulate Bcl-xL membrane interactions, including interactions with lipids and others members of the Bcl-2 family, and pH. However, the combined effect of these factors has not been studied systematically. In this study we have used different fluorescence-based methods to determine how different lipids and tBid modulate the pH-triggered membrane interactions of BCL-xL. FRET measurements between donor labeled protein and acceptor labeled LUVs were used to study membrane anchoring. Our results show that full-length BCL-xL is capable of anchoring to negatively charged bilayers at neutral pH, with higher levels observed under higher membrane electrostatic potential. The addition of tBid into the system caused a small reduction in the free energy of protonation of Bcl-xL membrane association. The pH-mediated insertion of Bcl-xL was monitored by placing the environment sensitive probe NBD at the middle of helix α6. We determined that the insertion/refolding of BCL-xL in the membrane follows the same lipid dependence trend as its initial anchoring step. In this case, however, interaction with tBid caused a much larger decrease on the free energy of protonation of Bcl-xL. We conclude that the tBid dependent modulation of BCL-xL activity is mostly performed in the membrane by preventing the formation of the membrane-inserted conformation of Bcl-xL, and less in restricting its membrane anchoring. We show that BCL-xL lipid interactions could be directly and indirectly controlled by changes in membrane lipid composition during apoptosis.

Moving along the Free Energy Landscape of Membrane Insertion of the Diphtheria Toxin Translocation Domain

The pH-triggered membrane insertion of the translocation (T) domain is critical for the entry of the diphtheria toxin into the target cell. Previously we characterized the kinetic pathway of membrane insertion of the T domain, which consists of a sequence of conformational changes that convert a water-soluble state into a transmembrane state. Here we utilize various thermodynamic approaches to determine the changes in the Gibbs free energy associated with these conformational changes. The initial conformational change, which occurs in solution, was studied by thermal and chemical denaturation using differential scanning calorimetry, circular dichroism, and fluorescence spectroscopy. We found that acidification of solution, which results in the formation of the membrane-competent form, reduces the thermodynamic stability of the T-domain by about 3-5 kcal/mol, depending on the experimental conditions. For thermodynamic studies of membrane insertion we applied a novel approach that combines fluorescence correlation spectroscopy with the use of fluorinated surfactants as chemical chaperones. We estimated that the free energy values for the transition from a membrane-competent state in solution to the interfacial intermediate, and to the final transmembrane state are about −8.2 ± 0.2 kcal/mol and −12.0 ± 0.2 kcal/mol, respectively. The free energy barrier between the two states is modulated by the presence of anionic lipids. We summarize our findings in a proposed free energy landscape for the refolding and bilayer insertion of the T domain.Supported by NIH GM-069783 (A.S.L.), and Fulbright-CONICYT and BRTP (M.V.U.)

Modulation of Membrane Interactions of Anti-Apoptotic Regulator Bcl-xL by Lipids

The Bcl-2 family of proteins (e.g., pro-apoptotic Bax and anti-apoptotic Bcl-xL) regulates the mitochondrial outer membrane permeabilization during the early stages of apoptosis. The prevalent Embedded Together Model of Bcl-2 action suggests that the membrane environment is critical for their proper functional interactions, consistent with the increasing evidence of lipids being involved in the regulation of apoptotic response. In this study, we apply a collection of fluorescence-based methods to investigate the effect of various lipids on the pH-triggered membrane interactions of Bcl-xL. The initial membrane association was studied using a FRET assay with donor-labeled Bcl-xL and acceptor-labeled vesicles, while the insertion/refolding of Bcl-xL into the membrane was monitored using the environment-sensitive probe NBD selectively attached in the middle of hydrophobic helix α6. Our results demonstrate that the lipid composition affects the pH-dependence of both initial membrane association and subsequent insertion/refolding of Bcl-xL. We found that a linear correlation exists between the membrane surface potential created by anionic lipids and the pKa of membrane binding, suggesting that the initial step is controlled by an electrostatic mechanism. The effect of lipids on the membrane insertion/refolding step is more complex and appears to be influenced by the size of the lipid headgroup. The kinetics of both the membrane association and membrane insertion/refolding is affected by the presence of non-bilayer forming lipids commonly found in mitochondria. While the presence of phosphatidylethanolamine accelerated the process, addition of lysophosphatidylcholine had the opposite effect, suggesting that mechanical properties of the bilayer also play a role. Taken together our results indicate that lipids can modulate the membrane interactions of Bcl-xL in multiple ways, providing an additional regulatory mechanism that ensures proper control of a complex cascade of apoptotic reactions leading to cell death or survival. NIHGM-069783, Fulbright-CONICYT, BRTP.