Beta-Barrel Channel Response to High Electric Fields: Functional Gating or Reversible Denaturation?

Ion channels exhibit gating behavior, fluctuating between open and closed states, with the transmembrane voltage serving as one of the essential regulators of this process. Voltage gating is a fundamental functional aspect underlying the regulation of ion-selective, mostly α-helical, channels primarily found in excitable cell membranes. In contrast, there exists another group of larger, and less selective, ß-barrel channels of a different origin, which are not directly associated with cell excitability. Remarkably, these channels can also undergo closing, or "gating", induced by sufficiently strong electric fields. Once the field is removed, the channels reopen, preserving a memory of the gating process. In this study, we explored the hypothesis that the voltage-induced closure of the ß-barrel channels can be seen as a form of reversible protein denaturation by the high electric fields applied in model membranes experiments-typically exceeding twenty million volts per meter-rather than a manifestation of functional gating. Here, we focused on the bacterial outer membrane channel OmpF reconstituted into planar lipid bilayers and analyzed various characteristics of the closing-opening process that support this idea. Specifically, we considered the nearly symmetric response to voltages of both polarities, the presence of multiple closed states, the stabilization of the open conformation in channel clusters, the long-term gating memory, and the Hofmeister effects in closing kinetics. Furthermore, we contemplate the evolutionary aspect of the phenomenon, proposing that the field-induced denaturation of membrane proteins might have served as a starting point for their development into amazing molecular machines such as voltage-gated channels of nerve and muscle cells.

Probing protein nanopores with poly(ethylene glycol)s.

Neutral water-soluble poly(ethylene glycol)s (PEGs) have been extensively explored in protein nanopore research for the past several decades. The principal use of PEGs is to investigate the membrane protein ion channel physical characteristics and transport properties. In addition, protein nanopores are used to study polymer-protein interactions and polymer physicochemical properties. In this review, we focus on the biophysical studies on probing protein ion channels with PEGs, specifically on nanopore sizing by PEG partitioning. We discuss the fluctuation analysis of ion channel currents in response to the PEGs moving within their confined geometries. The advantages, limitations, and recent developments of the approach are also addressed.

Anthrax toxin channel: What we know based on over 30 years of research.

Protective antigen channel is the central component of the deadly anthrax exotoxin responsible for binding and delivery of the toxin's enzymatic lethal and edema factor components into the cytosol. The channel, which is more than three times longer than the lipid bilayer membrane thickness and has a 6-Å limiting diameter, is believed to provide a sophisticated unfoldase and translocase machinery for the foreign protein transport into the host cell cytosol. The tripartite toxin can be reengineered, one component at a time or collectively, to adapt it for the targeted cancer therapeutic treatments. In this review, we focus on the biophysical studies of the protective antigen channel-forming activity, small ion transport properties, enzymatic factor translocation, and blockage comparing it with the related clostridial binary toxin channels. We address issues linked to the anthrax toxin channel structural dynamics and lipid dependence, which are yet to become generally recognized as parts of the toxin translocation machinery.

Hydrophobic Gating and 1/f Noise of the Anthrax Toxin Channel.

"Pink" or 1/f noise is a natural phenomenon omnipresent in physics, economics, astrophysics, biology, and even music and languages. In electrophysiology, the stochastic activity of a number of biological ion channels and artificial nanopores could be characterized by current noise with a 1/f power spectral density. In the anthrax toxin channel (PA63), it appears as fast voltage-independent current interruptions between conducting and nonconducting states. This behavior hampers potential development of PA63 as an ion-channel biosensor. On the bright side, the PA63 flickering represents a mesmerizing phenomenon to investigate. Notably, similar 1/f fluctuations are observed in the channel-forming components of clostridial binary C2 and iota toxins, which share functional and structural similarities with the anthrax toxin channel. Similar to PA63, they are evolved to translocate the enzymatic components of the toxins into the cytosol. Here, using high-resolution single-channel lipid bilayer experiments and all-atom molecular dynamic simulations, we suggest that the 1/f noise in PA63 occurs as a result of "hydrophobic gating" at the ϕ-clamp region, the phenomenon earlier observed in several water-filled channels "fastened" inside by the hydrophobic belts. The ϕ-clamp is a narrow "hydrophobic ring" in the PA63 lumen formed by seven or eight phenylalanine residues at position 427, conserved in the C2 and iota toxin channels, which catalyzes protein translocation. Notably, the 1/f noise remains undetected in the F427A PA63 mutant. This finding can elucidate the functional purpose of 1/f noise and its possible role in the transport of the enzymatic components of binary toxins.

Exploring the Nature of Cationic Blocker Recognition by the Anthrax Toxin Channel.

Obstructing conductive pathways of the channel-forming toxins with targeted blockers is a promising drug design approach. Nearly all tested positively charged ligands have been shown to reversibly block the cation-selective channel-forming protective antigen (PA63) component of the binary anthrax toxin. The cationic ligands with more hydrophobic surfaces, particularly those carrying aromatic moieties, inhibited PA63 more effectively. To understand the physical basis of PA63 selectivity for a particular ligand, detailed information is required on how the blocker structural elements (e.g., positively charged and aromatic groups) influence the molecular kinetics of the blocker/channel binding reactions. In this study, we address this problem using the high-resolution single-channel planar lipid bilayer technique. Several structurally distinct cationic blockers, namely per-6-S-(3-amino) propyl-ß-cyclodextrin, per-6-S-(3-aminomethyl) benzyl-α-cyclodextrin, per-6-S-(3-aminomethyl) benzyl-ß-cyclodextrin, per-6-S-(3-aminomethyl) benzyl-γ-cyclodextrin, methyltriphenylphosphonium ion, and G0 polyamidoamine dendrimer are tested for their ability to inhibit the heptameric and octameric PA63 variants and PA63F427A mutant. The F427 residues form a hydrophobic constriction region inside the channel, known as the "ϕ-clamp." We show that the cationic blockers interact with PA63 through a combination of forces. Analysis of the binding reaction kinetics suggests the involvement of cation-π, Coulomb, and salt-concentration-independent π-π or hydrophobic interactions in the cationic cyclodextrin binding. It is possible that these blockers bind to the ϕ-clamp and are also stabilized by the Coulomb interactions of their terminal amino groups with the water-exposed negatively charged channel residues. In PA63F427A, only the suggested Coulomb component of the cyclodextrin interaction remains. Methyltriphenylphosphonium ion and G0 polyamidoamine dendrimer, despite being positively charged, interact primarily with the ϕ-clamp. We also show that seven- and eightfold symmetric cyclodextrins effectively block the heptameric and octameric forms of PA63 interchangeably, adding flexibility to the earlier formulated blocker/target symmetry match requirement.

Effect of late endosomal DOBMP lipid and traditional model lipids of electrophysiology on the anthrax toxin channel activity.

Anthrax toxin action requires triggering of natural endocytic transport mechanisms whereby the binding component of the toxin forms channels (PA63) within endosomal limiting and intraluminal vesicle membranes to deliver the toxin's enzymatic components into the cytosol. Membrane lipid composition varies at different stages of anthrax toxin internalization, with intraluminal vesicle membranes containing ~70% of anionic bis(monoacylglycero)phosphate lipid. Using model bilayer measurements, we show that membrane lipids can have a strong effect on the anthrax toxin channel properties, including the channel-forming activity, voltage-gating, conductance, selectivity, and enzymatic factor binding. Interestingly, the highest PA63 insertion rate was observed in bis(monoacylglycero)phosphate membranes. The molecular dynamics simulation data show that the conformational properties of the channel are different in bis(monoacylglycero)phosphate compared to PC, PE, and PS lipids. The anthrax toxin protein/lipid bilayer system can be advanced as a novel robust model to directly investigate lipid influence on membrane protein properties and protein/protein interactions.

Effect of endosomal acidification on small ion transport through the anthrax toxin PA63 channel.

Tight regulation of pH is critical for the structure and function of cells and organelles. The pH environment changes dramatically along the endocytic pathway, an internalization transport process that is 'hijacked' by many intracellularly active bacterial exotoxins, including the anthrax toxin. Here, we investigate the role of pH on single-channel properties of the anthrax toxin protective antigen (PA63 ). Using conductance and current noise analysis, blocker binding, ion selectivity, and poly(ethylene glycol) partitioning measurements, we show that the channel exists in two different open states ('maximum' and 'main') at pH ≥ 5.5, while only a maximum conductance state is detected at pH < 5.5. We describe two substantially distinct patterns of PA63 conductance dependence on KCl concentration uncovered at pH 6.5 and 4.5.

Multivalent Inhibitors of Channel-Forming Bacterial Toxins.

Rational design of multivalent molecules represents a remarkable modern tool to transform weak non-covalent interactions into strong binding by creating multiple finely-tuned points of contact between multivalent ligands and their supposed multivalent targets. Here, we describe several prominent examples where the multivalent blockers were investigated for their ability to directly obstruct oligomeric channel-forming bacterial exotoxins, such as the pore-forming bacterial toxins and B component of the binary bacterial toxins. We address problems related to the blocker/target symmetry match and nature of the functional groups, as well as chemistry and length of the linkers connecting the functional groups to their multivalent scaffolds. Using the anthrax toxin and AB5 toxin case studies, we briefly review how the oligomeric toxin components can be successfully disabled by the multivalent non-channel-blocking inhibitors, which are based on a variety of multivalent scaffolds.

Impact of Dendrimer Terminal Group Chemistry on Blockage of the Anthrax Toxin Channel: A Single Molecule Study.

Nearly all the cationic molecules tested so far have been shown to reversibly block K⁺ current through the cation-selective PA63 channels of anthrax toxin in a wide nM-mM range of effective concentrations. A significant increase in channel-blocking activity of the cationic compounds was achieved when multiple copies of positively charged ligands were covalently linked to multivalent scaffolds, such as cyclodextrins and dendrimers. Even though multivalent binding can be strong when the individual bonds are relatively weak, for drug discovery purposes we often strive to design multivalent compounds with high individual functional group affinity toward the respective binding site on a multivalent target. Keeping this requirement in mind, here we perform a single-channel/single-molecule study to investigate kinetic parameters of anthrax toxin PA63 channel blockage by second-generation (G2) poly(amido amine) (PAMAM) dendrimers functionalized with different surface ligands, including G2-NH2, G2-OH, G2-succinamate, and G2-COONa. We found that the previously reported difference in IC50 values of the G2-OH/PA63 and G2-NH2/PA63 binding was determined by both on- and off-rates of the reversible dendrimer/channel binding reaction. In 1 M KCl, we observed a decrease of about three folds in k o n and a decrease of only about ten times in t r e s with G2-OH compared to G2-NH2. At the same time for both blockers, k o n and t r e s increased dramatically with transmembrane voltage increase. PAMAM dendrimers functionalized with negatively charged succinamate, but not carboxyl surface groups, still had some residual activity in inhibiting the anthrax toxin channels. At 100 mV, the on-rate of the G2-succinamate binding was comparable with that of G2-OH but showed weaker voltage dependence when compared to G2-OH and G2-NH2. The residence time of G2-succinamate in the channel exhibited opposite voltage dependence compared to G2-OH and G2-NH2, increasing with the cis-negative voltage increase. We also describe kinetics of the PA63 ion current modulation by two different types of the "imperfect" PAMAM dendrimers, the mixed-surface G2 75% OH 25% NH2 dendrimer and G3-NH2 dendron. At low voltages, both "imperfect" dendrimers show similar rate constants but significantly weaker voltage sensitivity when compared with the intact G2-NH2 PAMAM dendrimer.

Inhibiting bacterial toxins by channel blockage.

Emergent rational drug design techniques explore individual properties of target biomolecules, small and macromolecule drug candidates, and the physical forces governing their interactions. In this minireview, we focus on the single-molecule biophysical studies of channel-forming bacterial toxins that suggest new approaches for their inhibition. We discuss several examples of blockage of bacterial pore-forming and AB-type toxins by the tailor-made compounds. In the concluding remarks, the most effective rationally designed pore-blocking antitoxins are compared with the small-molecule inhibitors of ion-selective channels of neurophysiology.

Channel-forming bacterial toxins in biosensing and macromolecule delivery.

To intoxicate cells, pore-forming bacterial toxins are evolved to allow for the transmembrane traffic of different substrates, ranging from small inorganic ions to cell-specific polypeptides. Recent developments in single-channel electrical recordings, X-ray crystallography, protein engineering, and computational methods have generated a large body of knowledge about the basic principles of channel-mediated molecular transport. These discoveries provide a robust framework for expansion of the described principles and methods toward use of biological nanopores in the growing field of nanobiotechnology. This article, written for a special volume on "Intracellular Traffic and Transport of Bacterial Protein Toxins", reviews the current state of applications of pore-forming bacterial toxins in small- and macromolecule-sensing, targeted cancer therapy, and drug delivery. We discuss the electrophysiological studies that explore molecular details of channel-facilitated protein and polymer transport across cellular membranes using both natural and foreign substrates. The review focuses on the structurally and functionally different bacterial toxins: gramicidin A of Bacillus brevis, α-hemolysin of Staphylococcus aureus, and binary toxin of Bacillus anthracis, which have found their "second life" in a variety of developing medical and technological applications.

Tailored cyclodextrin pore blocker protects mammalian cells from clostridium difficile binary toxin CDT.

Some Clostridium difficile strains produce, in addition to toxins A and B, the binary toxin Clostridium difficile transferase (CDT), which ADP-ribosylates actin and may contribute to the hypervirulence of these strains. The separate binding and translocation component CDTb mediates transport of the enzyme component CDTa into mammalian target cells. CDTb binds to its receptor on the cell surface, CDTa assembles and CDTb/CDTa complexes are internalised. In acidic endosomes, CDTb mediates the delivery of CDTa into the cytosol, most likely by forming a translocation pore in endosomal membranes. We demonstrate that a seven-fold symmetrical positively charged ß-cyclodextrin derivative, per-6-S-(3-aminomethyl)benzylthio-ß-cyclodextrin, which was developed earlier as a potent inhibitor of the translocation pores of related binary toxins of Bacillus anthracis, Clostridium botulinum and Clostridium perfringens, protects cells from intoxication with CDT. The pore blocker did not interfere with the CDTa-catalyzed ADP-ribosylation of actin or toxin binding to Vero cells but inhibited the pH-dependent membrane translocation of CDTa into the cytosol. In conclusion, the cationic ß-cyclodextrin could serve as the lead compound in a development of novel pharmacological strategies against the CDT-producing strains of C. difficile.

Cationic PAMAM dendrimers as pore-blocking binary toxin inhibitors.

Dendrimers are unique highly branched macromolecules with numerous groundbreaking biomedical applications under development. Here we identified poly(amido amine) (PAMAM) dendrimers as novel blockers for the pore-forming B components of the binary anthrax toxin (PA63) and Clostridium botulinum C2 toxin (C2IIa). These pores are essential for delivery of the enzymatic A components of the internalized toxins from endosomes into the cytosol of target cells. We demonstrate that at low µM concentrations cationic PAMAM dendrimers block PA63 and C2IIa to inhibit channel-mediated transport of the A components, thereby protecting HeLa and Vero cells from intoxication. By channel reconstitution and high-resolution current recording, we show that the PAMAM dendrimers obstruct transmembrane PA63 and C2IIa pores in planar lipid bilayers at nM concentrations. These findings suggest a new potential role for the PAMAM dendrimers as effective polyvalent channel-blocking inhibitors, which can protect human target cells from intoxication with binary toxins from pathogenic bacteria.

Designing inhibitors of anthrax toxin.

INTRODUCTION: Present-day rational drug design approaches are based on exploiting unique features of the target biomolecules, small- or macromolecule drug candidates and physical forces that govern their interactions. The 2013 Nobel Prize in chemistry awarded 'for the development of multiscale models for complex chemical systems' once again demonstrated the importance of the tailored drug discovery that reduces the role of the trial-and-error approach to a minimum. The intentional dissemination of Bacillus anthracis spores in 2001 via the so-called anthrax letters has led to increased efforts, politically and scientifically, to develop medical countermeasures that will protect people from the threat of anthrax bioterrorism. AREAS COVERED: This article provides an overview of the recent rational drug design approaches for discovering inhibitors of anthrax toxin. The review also directs the readers to the vast literature on the recognized advances and future possibilities in the field. EXPERT OPINION: Existing options to combat anthrax toxin lethality are limited. With the only anthrax toxin inhibiting therapy (protective antigen-targeting with a monoclonal antibody, raxibacumab) approved to treat inhalational anthrax, the situation, in our view, is still insecure. Further, the FDA's animal rule for drug approval, which clears compounds without validated efficacy studies on humans, creates a high level of uncertainty, especially when a well-characterized animal model does not exist. Better identification and validation of anthrax toxin therapeutic targets at the molecular level as well as elucidation of the parameters determining the corresponding therapeutic windows are still necessary for more effective therapeutic options.

Engineering anthrax toxin variants that exclusively form octamers and their application to targeting tumors.

Anthrax toxin protective antigen (PA) delivers its effector proteins into the host cell cytosol through formation of an oligomeric pore, which can assume heptameric or octameric states. By screening a highly directed library of PA mutants, we identified variants that complement each other to exclusively form octamers. These PA variants were individually nontoxic and demonstrated toxicity only when combined with their complementary partner. We then engineered requirements for activation by matrix metalloproteases and urokinase plasminogen activator into two of these variants. The resulting therapeutic toxin specifically targeted cells expressing both tumor associated proteases and completely stopped tumor growth in mice when used at a dose far below that which caused toxicity. This scheme for obtaining intercomplementing subunits can be employed with other oligomeric proteins and potentially has wide application.

Kinetics and thermodynamics of binding reactions as exemplified by anthrax toxin channel blockage with a cationic cyclodextrin derivative.

The thermodynamics of binding reactions is usually studied in the framework of the linear van't Hoff analysis of the temperature dependence of the equilibrium constant. The logarithm of the equilibrium constant is plotted versus inverse temperature to discriminate between two terms: an enthalpic contribution that is linear in the inverse temperature, and a temperature-independent entropic contribution. When we apply this approach to a particular case-blockage of the anthrax PA(63) channel by a multicharged cyclodextrin derivative-we obtain a nearly linear behavior with a slope that is characterized by enthalpy of about 1 kcal/mol. In contrast, from blocker partitioning between the channel and the bulk, we estimate the depth of the potential well for the blocker in the channel to be at least 8 kcal/mol. To understand this apparent discrepancy, we use a simple model of particle interaction with the channel and show that this significant difference between the two estimates is due to the temperature dependence of the physical forces between the blocker and the channel. In particular, we demonstrate that if the major component of blocker-channel interaction is van der Waals interactions and/or Coulomb forces in water, the van't Hoff enthalpy of the binding reaction may be close to zero or even negative, including cases of relatively strong binding. The results are quite general and, therefore, of importance for studies of enzymatic reactions, rational drug design, small-molecule binding to proteins, protein-protein interactions, and protein folding, among others.

Interactions of high-affinity cationic blockers with the translocation pores of B. anthracis, C. botulinum, and C. perfringens binary toxins.

Cationic ß-cyclodextrin derivatives were recently introduced as highly effective, potentially universal blockers of three binary bacterial toxins: anthrax toxin of Bacillus anthracis, C2 toxin of Clostridium botulinum, and iota toxin of Clostridium perfringens. The binary toxins are made of two separate components: the enzymatic A component, which acts on certain intracellular targets, and the binding/translocation B component, which forms oligomeric channels in the target cell membrane. Here we studied the voltage and salt dependence of the rate constants of binding and dissociation reactions of two structurally different ß-cyclodextrins (AmPrßCD and AMBnTßCD) in the PA(63), C2IIa, and Ib channels (B components of anthrax, C2, and iota toxins, respectively). With all three channels, the blocker carrying extra hydrophobic aromatic groups on the thio-alkyl linkers of positively charged amino groups, AMBnTßCD, demonstrated significantly stronger binding compared with AmPrßCD. This effect is seen as an increased residence time of the blocker in the channels, whereas the time between blockages characterizing the binding reaction on-rate stays practically unchanged. Surprisingly, the voltage sensitivity, expressed as a slope of the logarithm of the blocker residence time as a function of voltage, turned out to be practically the same for all six cases studied, suggesting structural similarities among the three channels. Also, the more-effective AMBnTßCD blocker shows weaker salt dependence of the binding and dissociation rate constants compared with AmPrßCD. By estimating the relative contributions of the applied transmembrane field, long-range Coulomb, and salt-concentration-independent, short-range forces, we found that the latter represent the leading interaction, which accounts for the high efficiency of blockage. In a search for the putative groups in the channel lumen that are responsible for the short-range forces, we performed measurements with the F427A mutant of PA(63), which lacks the functionally important phenylalanine clamp. We found that the on-rates of the blockage were virtually conserved, but the residence times and, correspondingly, the binding constants dropped by more than an order of magnitude, which also reduced the difference between the efficiencies of the two blockers.