Transmembrane Pore Research Articles

De novo design of peptides that form transmembrane barrel pores killing antibiotic resistant bacteria.

De novo design of peptides that self-assemble into transmembrane barrel-like nanopores is challenging due to the complexity of several competing interactions involving peptides, lipids, water, and ions. We have develop a computational approach for the de novo design of α-helical peptides that self-assemble into stable transmembrane barrel pores with a central nano-sized functional channel. We formulate the previously missing design guidelines and report 52 sequence patterns that can be tuned for specific applications using the identified role of each residue. Atomic force microscopy, fluorescent dye leakage, and cryo-EM experiments confirm that the designed peptides form leaky membrane nanopores in vitro. Customized designed peptides act as antimicrobial agents able to kill even antibiotic-resistant ESKAPE bacteria at micromolar concentrations, while exhibiting low toxicity to human cells. The peptides and their assembled nanopore structures can be similarly fine-tuned for other medical and biotechnological applications.

Uncovering individual protonation events in Kir2 channels.

Inwardly rectifying potassium (Kir) channels play a critical role in stabilizing the membrane potential, thus controlling numerous physiological phenomena in various tissues. Channel conductance can be activated by cytoplasmic modulators that open the channel at the ‘helix bundle crossing’ (HBC), formed by the coming together of the M2 helix from each of the four subunits, at the cytoplasmic end of the transmembrane pore.

Tuning the size and solvent-exposed hydrophobicity of a pore-forming peptide eliminates its cytotoxicity using a natural product.

Pore-forming toxins (PFTs) are a diverse group of peptides and proteins that interact with the cell membrane to form transmembrane pores which lead to to ion dyshomeostasis and cell death. Our previous work showed that claramine, an aminosterol derivative of cholesterol, protects brain and kidney cells from prototypical PFTs. This effect was not induced by a change in protein structure, but instead by incorporating into and changing the physico-chemical properties of the plasma membrane to prevent the binding of these toxins. In the search for compounds that can target the structures of PFTs directly to eliminate their toxicity, we found that the polyphenol epigallocatechin gallate (EGCG) directly interacts with the monomeric PFT melittin, the active component of honeybee venom, resulting in the elimination of its binding to the cell membrane and neutralization of its cytotoxicity. EGCG achieved this effect by lowering the solvent-exposed hydrophobicity of melittin and promoting its oligomerization into larger aggregates. These biophysical properties have also shown to play an important role in the membrane binding and associated cytotoxicity of misfolded protein oligomers implicit in neurodegenerative diseases. Our results show that this relationship translates to the attenuation of melittin cytotoxicity by EGCG. These findings emphasize the importance of the physico-chemical properties of PFTs in controlling their binding to cell membranes and suggest a potential mechanism for therapeutic development against PFTs.

Cryo-EM structures of the TolQ membrane protein in complex with inner membrane protein partner TolR from the opportunistic pathogen Acinetobacter baumannii provide information on structure and stoichiometry.

TolQ-TolR-TolA is an energy-transducing inner-membrane sub-complex conserved across Gram-negative bacteria. It maintains connection with periplasmic TolB-Pal sub-complex in an energy-dependent manner through TolA protein spanning the periplasm. Utilizing proton motive force (PMF), Tol-Pal complex contributes to the integrity of outer membrane and the whole cell envelope. TolQ remains the only part of Tol-Pal complex with unknown structure up to now. To address this, we determined 3.02 and 3.34 Å cryo-electron microscopy (cryo-EM) structures of the integral inner-membrane protein TolQ and in complex with TolR, respectively from Acinetobacter baumannii. A. baumannii is one of the most dreaded and clinically important Gram-negative pathogens causing a broad-spectrum nosocomial infection mainly among debilitated patients admitted to intensive care units. Our structural analysis revealed that TolQ forms a pentamer with a channel-like hydrophobic trans-membrane pore slightly shifted above the membrane opening into a large negatively charged cytoplasmic chamber which is in common with the closest TolQ-TolR paralogs ExbB-ExbD and MotA-MotB. We validated observed structural features of TolQ by prior functional data. According to our analysis the most functionally detrimental point mutations mapped on structure are localized inside the channel. Obtained structures suggest that two trans-membrane domains of TolR dimer fit inside TolQ channel in TolQ-TolR complex forming a proton-flow pathway for PMF utilization. Cryo-EM structures solved in this work were funded in whole or in part with Refeyn Travel Grant and U.S. federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under contract No. HHSN272201700060C (Center for Structural Genomics of Infectious Diseases (CSGID); http://csgid.org).

Antifungal efficacy of kenaf seed peptides mixture in cheese, safety assessment and unravelling its action mechanism against food spoilage fungi

This study investigated the preservation efficacy of kenaf seed peptides mixture (KSPM) in extending the shelf-life of cream cheese matrix and the effect on its physicochemical properties. Also, the safety of KSPM towards mammalian cells and the underlying fungicidal mode of action in-vitro and in-silico were assessed. The KSPM at 500 and 1000 mg/kg effectively prolonged the shelf-life of cream cheese for up to 11–21 days when stored at 25 °C and 4 °C, and significantly reduced the inoculated conidia counts by ∼3 log10 conidia/g. The safety assessment showed that KSPM maintained the proliferation of normal cell lines up to 30 mg/mL. The cell surface potential energy and hydrophobicity of fungi were altered due to the potent interactions between the peptides and the cell envelope, with KSPM causing significant fungal cell wall permeability and subsequent disruption of the cytoplasmic membrane integrity. KSPM treatment induced morphological destruction of conidia and mycelium and caused a significant loss of fungal cellular materials including K+, sugar, protein and nucleic acids (DNA and RNA), indicating irreversible damage to the fungal cytoplasmic membrane. The in-silico molecular docking study revealed that the peptides mixture efficiently interacted with fungal mannoprotein via the formation of hydrogen bonding, electrostatic interactions and hydrophobic forces. The molecular dynamics simulation showed that the peptide KPTGMR was tightly embedded into the fungal phospholipid bilayer membrane and inserted into the phosphate head groups which led to the formation of gaps, suggesting a transmembrane pore or channel action mechanism.

Pyroptosis Induction and Visualization at the Single-Cell Level Using Optogenetics.

Pyroptosis has been identified as a pro-inflammatory form of programmed cell death. It can be triggered by different stimuli including pathogen invasion or cell stress/danger signals releasing hundreds of proteins upon lysis that cause complex responses in neighboring cells. Pyroptosis is executed by the gasdermin (GSDM) family of proteins which, upon cleavage by caspases, form transmembrane pores that release cytokines to induce inflammation. However, despite the importance of gasdermins in the development of inflammatory diseases and cancer, a lot is still to be understood in the downstream consequences of this cell death pathway. Currently, conventional methods, such as drug treatments or chemically forced oligomerization, are limited in the spatiotemporal analysis of pyroptosis signaling in the cellular population, since all cells are primed for undergoing pyroptosis. Here, we provide a protocol for the application of a novel optogenetics tool called NLS_PhoCl_N-GSDMD_mCherry that enables precise temporal and spatial pyroptosis induction in a confocal microscopy setup, followed by imaging of the cell death process and subsequent quantitative analysis of the experiment. This tool opens new opportunities for the study of pyroptosis activation and of its effects on the bystander cell responses.

Monitoring Calcium Fluxes and Lysosome Exocytosis During Pyroptosis.

Inflammasome-mediated activation of inflammatory caspases (caspase-1, caspase-4, caspase-5, caspase-11) initiates a cascade of cellular events that lead to proinflammatory cell death, or pyroptosis. Proteolytic cleavage of gasdermin D results in the formation of transmembrane pores that allow the release of mature cytokines IL-1β and IL-18. Gasdermin pores also allow calcium influx through the plasma membrane, triggering the fusion of lysosomal compartments with the cell surface and release of their contents into the extracellular milieu in a process termed lysosome exocytosis. This chapter outlines methods for measuring calcium flux, lysosome exocytosis, and membrane disruption after inflammatory caspase activation.

Cells Responding to Closely Related Cholesterol-Dependent Cytolysins Release Extracellular Vesicles with a Common Proteomic Content Including Membrane Repair Proteins.

The plasma membrane (PM) protects cells from extracellular threats and supports cellular homeostasis. Some pathogens produce pore-forming toxins (PFTs) that disrupt PM integrity by forming transmembrane pores. High PFT concentrations cause massive damage leading to cell death and facilitating infection. Sub-lytic PFT doses activate repair mechanisms to restore PM integrity, support cell survival and limit disease. Shedding of extracellular vesicles (EVs) has been proposed as a key mechanism to eliminate PFT pores and restore PM integrity. We show here that cholesterol-dependent cytolysins (CDCs), a specific family of PFTs, are at least partially eliminated through EVs release, and we hypothesize that proteins important for PM repair might be included in EVs shed by cells during repair. To identify new PM repair proteins, we collected EVs released by cells challenged with sub-lytic doses of two different bacterial CDCs, listeriolysin O and pneumolysin, and determined the EV proteomic repertoire by LC-MS/MS. Intoxicated cells release similar EVs irrespectively of the CDC used. Also, they release more and larger EVs than non-intoxicated cells. A cluster of 70 proteins including calcium-binding proteins, molecular chaperones, cytoskeletal, scaffold and membrane trafficking proteins, was detected enriched in EVs collected from intoxicated cells. While some of these proteins have well-characterized roles in repair, the involvement of others requires further study. As proof of concept, we show here that Copine-1 and Copine-3, proteins abundantly detected in EVs released by intoxicated cells, are required for efficient repair of CDC-induced PM damage. Additionally, we reveal here new proteins potentially involved in PM repair and give new insights into common mechanisms and machinery engaged by cells in response to PM damage.

Mutational Insight into Allosteric Regulation of Kir Channel Activity.

Potassium (K+) channels are regulated in part by allosteric communication between the helical bundle crossing, or inner gate, and the selectivity filter, or outer gate. This network is triggered by gating stimuli. In concert, there is an allosteric network which is a conjugated set of interactions which correlate long-range structural rearrangements necessary for channel function. Inward-rectifier K+ (Kir) channels favor inward K+ conductance, are ligand-gated, and help establish resting membrane potentials. KirBac1.1 is a bacterial Kir (KirBac) channel homologous to human Kir (hKir) channels. Additionally, KirBac1.1 is gated by the anionic phospholipid ligand phosphatidylglycerol (PG). In this study, we use site-directed mutagenesis to investigate residues involved in the KirBac1.1 gating mechanism and allosteric network we previously proposed using detailed solid-state NMR (SSNMR) measurements. Using fluorescence-based K+ and sodium (Na+) flux assays, we identified channel mutants with impaired function that do not alter selectivity of the channel. In tandem, we performed coarse grain molecular dynamics simulations, observing changes in PG-KirBac1.1 interactions correlated with mutant channel activity and contacts between the two transmembrane helices and pore helix tied to this behavior. Lipid affinity is closely tied to the proximity of two tryptophan residues on neighboring subunits which lure anionic lipids to a cationic pocket formed by a cluster of arginine residues. Thus, these simulations establish a structural and functional basis for the role of each mutated site in the proposed allosteric network. The experimental and simulated data provide insight into key functional residues involved in gating and lipid allostery of K+ channels. Our findings also have direct implications on the physiology of hKir channels due to conservation of many of the residues identified in this work from KirBac1.1.

Genetic and pharmacological targeting of GSDMD ameliorates systemic inflammation in macrophage activation syndrome

Macrophage activation syndrome (MAS), a potentially life-threatening complication of autoimmune/autoinflammatory diseases, is characterized by the excessive expansion and activation of macrophages and cytotoxic T lymphocytes in multiple organs. Most commonly, MAS occurs in patients with systemic juvenile idiopathic arthritis and in its adult equivalent, adult-onset Still's disease (AOSD). Gasdermin D (GSDMD) is a critical pore-forming effector protein that mediates pro-inflammatory cytokine secretion via releasing its N terminal fragments to form transmembrane pores. GSDMD has been implicated in various inflammatory diseases, however, its role in MAS remains elusive. Here, we unveiled that the serum levels of GSDMD-N were elevated in patients with AOSD compared to heathy controls. In addition, the emergence of MAS features in AOSD patients resulted in further elevation. The serum levels of GSDMD were positively correlated with ferritin and interleukin-18 (IL-18). Repeated toll-like receptor 9 stimulation with unmethylated cytosine-phosphate-guanine (CpG) induced MAS symptoms in wild-type mice, including body weight loss, pancytopenia and hepatosplenomegaly. Genetic deletion and pharmacological inhibition of GSDMD ameliorated MAS symptoms in mice with the concomitant reduction of splenic and hepatic macrophage infiltration and IL-18 production. Consistent with these in vivo results, bone marrow-derived macrophages obtained from GSDMD−/− mice or treated with GSDMD inhibitor disulfiram exhibited attenuated IL-18 expression after CpG stimulation. Collectively, our findings identified GSDMD as a novel marker for MAS complication and a promising target for MAS treatment.

The human TRPA1 intrinsic cold and heat sensitivity involves separate channel structures beyond the N-ARD domain

TRP channels sense temperatures ranging from noxious cold to noxious heat. Whether specialized TRP thermosensor modules exist and how they control channel pore gating is unknown. We studied purified human TRPA1 (hTRPA1) truncated proteins to gain insight into the temperature gating of hTRPA1. In patch-clamp bilayer recordings, ∆1–688 hTRPA1, without the N-terminal ankyrin repeat domain (N-ARD), was more sensitive to cold and heat, whereas ∆1–854 hTRPA1, also lacking the S1–S4 voltage sensing-like domain (VSLD), gained sensitivity to cold but lost its heat sensitivity. In hTRPA1 intrinsic tryptophan fluorescence studies, cold and heat evoked rearrangement of VSLD and the C-terminus domain distal to the transmembrane pore domain S5–S6 (CTD). In whole-cell electrophysiology experiments, replacement of the CTD located cysteines 1021 and 1025 with alanine modulated hTRPA1 cold responses. It is proposed that hTRPA1 CTD harbors cold and heat sensitive domains allosterically coupled to the S5–S6 pore region and the VSLD, respectively.

Label-free high-precise nanopore detection of endopeptidase activity of anthrax lethal factor regulated by diverse conditions

Endopeptidase activity of anthrax lethal factor (aLF) prevents the destroy of anthracis spore intracellularly by host macrophages, meanwhile disables the signaling pathways extracellularly that leads to host lethality. Hence, inhibitory of this activity is expected to be an alternative option to cure anthrax infection. Herein, we fabricated a nanopore platform via transmembrane pore construction in vitro, which allows precise mimics, monitoring of intercellular proteinic transport and enables the quantitative detection of aLF endopeptidase activity towards MAPKK signaling protein at single molecule level. Next, we inhibited the aLF activity via screening approaches of protein-metal ion acquisition and other condition controlment (proton/hydroxide strength, adapted temperature, ionizing irradiation), which were identified by nanopore electrokinetic study. Upon the results, we found that Ca2+, Mg2+, Mn2+, Ni2+ collaborating with Zn2+ promote aLF activity efficiently. In contrary, Cd2+, Co2+, Cu2+ have great inhibitory effect. Result further revealed that, the speed of aLF endopeptidase activity with different ions functions as the nanopore signal frequency in linear manner, which enables evident distinction of those divalent ions using this proteinase assay. We also found the higher strength of the proton or hydroxide, the higher the inhibitory to aLF activity. Besides, adapted temperature and γ-ray also play integral roles in inhibiting this activity. Our results lay experimental basis for accurate detection of aLF activity, meanwhile provide new direction to screening novel stimuli-responsive inhibitors specific to aLF.

Self-Assembly of Linear, Natural Antimicrobial Peptides: An Evolutionary Perspective.

Antimicrobial peptides are an ancient and innate system of host defence against a wide range of microbial assailants. Mechanistically, unstructured peptides undergo a secondary structure transition into amphipathic α-helices, upon contact with membrane surfaces. This leads to peptide binding and removal of the membrane components in a detergent-like manner or via self-organisation into trans-membrane pores (either barrel-stave or toroidal pore) thereby destroying the microbe. Self-assembly of antimicrobial peptides into oligomers and ultimately amyloid has been mostly examined in parallel, however recent findings link diseases, such as Alzheimer's disease as an aberrant activity of a protective neuropeptide with antimicrobial activity. These self-assembled oligomers can also interact with membranes. Here, we review those antimicrobial peptides reported to self-assemble into amyloid, where supported by structural evidence. We consider their membrane activities as antimicrobial peptides and present evidence of consistent self-assembly patterns across major evolutionary groups. Trends are apparent across these groups, supporting the mounting data that self-assembly of antimicrobial peptides into amyloid should be considered as synergistic to the antimicrobial peptide response.

Three dimensional modelling of mutant Kir2.1 channel PIP2 interactions help stratify arrhythmia severity in Andersen Tawil syndrome type 1

Abstract Background Andersen-Tawil type 1 (ATS1) is associated with loss-of-function mutations in the inward rectifier potassium channel Kir2.1, which controls cardiac excitability and impulse conduction. Phosphatidylinositol-4,5-bisphosphate (PIP2) acts as an essential cofactor regulating the opening of Kir2.1 channels. Fifty percent of reported ATS1 mutations affect Kir2.1-PIP2 interactions, leading to ECG defects, ventricular arrhythmias and sudden cardiac death (SCD) by mechanisms that are poorly understood. Purpose To test the hypothesis that the degree of arrhythmogenic severity of ATS1 mutations disrupting PIP2-Kir2.1 binding may be predicted by the level of polarization of the mutant Kir2.1 channel pore. Methods We first used a statistical mean value approach to classify the 40 known arrhythmogenic ATS1 mutations impacting Kir2.1-PIP2 interaction (N=260 individuals) according to arrhythmogenic severity, ranging from SCD through ventricular bigeminy and QT prolongation. We then generated 3D in-silico atomic models of the wildtype channel and the 10 mutant channels with the most severe arrhythmic phenotype to assess the mechanism of the structural defects associated with Kir2.1-PIP2 disruption. Results Our cardiac lethality scoring stratifying Kir2.1 mutations according to arrhythmogenic severity was validated by three additional biostatical quantitative measures. On in-silico modelling, wildtype Kir2.1 channels without PIP2 binding had transmembrane and cytoplasmic pore radius of 1.5 and 3 Å, respectively. Kir2.1-PIP2 interactions increased transmembrane and cytoplasmic pore radius to 3 and 6 Å, respectively. All 10 Kir2.1 mutations had similar transmembrane and cytoplasmic pore radius of ∼1.0 and ∼3.0 Å, respectively. The most severe mutations yielded pore channels with highly polarized electrostatic forces. Remarkably, simulations showed a descending electrostatic pattern at the transmembrane region of PIP2 binding, where the more severe the mutation, the more positive that region was. Structural changes produced by mutations correlated with cardiac severity (R2=0.51; p<0.005) in that the most drastically altered protein structure correlated with the most severe arrhythmic phenotype. Conclusions Computer simulations of mutant Kir2.1 channel structure from the most arrhythmogenic to the least arrhythmogenic predict a gradual decrease in polarization of electrostatic forces along the Kir2.1 channel pore. The results reveal a novel mechanistic stratification of arrhythmogenic severity of ATS1 mutant Kir2.1 channel-PIP2 interactions and open new pathways for developing more personalized ATS1 patient therapies. Funding Acknowledgement Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): La Caixa Banking Foundation under the project code HR18-00304Fundaciόn La Marato TV3: Ayudas a la investigaciόn en enfermedades raras 2020

Assembly of transmembrane pores from mirror-image peptides

Tailored transmembrane alpha-helical pores with desired structural and functional versatility have promising applications in nanobiotechnology. Herein, we present a transmembrane pore DpPorA, based on the natural pore PorACj, built from D-amino acid α-helical peptides. Using single-channel current recordings, we show that DpPorA peptides self-assemble into uniform cation-selective pores in lipid membranes and exhibit properties distinct from their L-amino acid counterparts. DpPorA shows resistance to protease and acts as a functional nanopore sensor to detect cyclic sugars, polypeptides, and polymers. Fluorescence imaging reveals that DpPorA forms well-defined pores in giant unilamellar vesicles facilitating the transport of hydrophilic molecules. A second D-amino acid peptide based on the polysaccharide transporter Wza forms transient pores confirming sequence specificity in stable, functional pore formation. Finally, molecular dynamics simulations reveal the specific alpha-helical packing and surface charge conformation of the D-pores consistent with experimental observations. Our findings will aid the design of sophisticated pores for single-molecule sensing related technologies.

A Novel Cryptic Clostridial Peptide That Kills Bacteria by a Cell Membrane Permeabilization Mechanism.

ABSTRACTThis work reports detailed characteristics of the antimicrobial peptide Intestinalin (P30), which is derived from the LysC enzyme of Clostridium intestinale strain URNW. The peptide shows a broader antibacterial spectrum than the parental enzyme, showing potent antimicrobial activity against clinical strains of Gram-positive staphylococci and Gram-negative pathogens and causing between 3.04 ± 0.12 log kill for Pseudomonas aeruginosa PAO1 and 7.10 ± 0.05 log kill for multidrug-resistant Acinetobacter baumannii KPD 581 at a 5 μM concentration. Moreover, Intestinalin (P30) prevents biofilm formation and destroys 24-h and 72-h biofilms formed by Acinetobacter baumannii CRAB KPD 205 (reduction levels of 4.28 and 2.62 log CFU/mL, respectively). The activity of Intestinalin is combined with both no cytotoxicity and little hemolytic effect against mammalian cells. The nuclear magnetic resonance and molecular dynamics (MD) data show a high tendency of Intestinalin to interact with the bacterial phospholipid cell membrane. Although positively charged, Intestinalin resides in the membrane and aggregates into small oligomers. Negatively charged phospholipids stabilize peptide oligomers to form water- and ion-permeable pores, disrupting the integrity of bacterial cell membranes. Experimental data showed that Intestinalin interacts with negatively charged lipoteichoic acid (logK based on isothermal titration calorimetry, 7.45 ± 0.44), causes membrane depolarization, and affects membrane integrity by forming large pores, all of which result in loss of bacterial viability.IMPORTANCE Antibiotic resistance is rising rapidly among pathogenic bacteria, becoming a global public health problem that threatens the effectiveness of therapies for many infectious diseases. In this respect, antimicrobial peptides appear to be an interesting alternative to combat bacterial pathogens. Here, we report the characteristics of an antimicrobial peptide (of 30 amino acids) derived from the clostridial LysC enzyme. The peptide showed killing activity against clinical strains of Gram-positive and Gram-negative pathogens. Experimental data and computational modeling showed that this peptide forms transmembrane pores, directly engaging the negatively charged phospholipids of the bacterial cell membrane. Consequently, dissipation of the electrochemical gradient across cell membranes affects many vital processes, such as ATP synthesis, motility, and transport of nutrients. This kind of dysfunction leads to the loss of bacterial viability. Our firm conviction is that the presented study will be a helpful resource in searching for novel antimicrobial peptides that could have the potential to replace conventional antibiotics.

Membranolytic Mechanism of Amphiphilic Antimicrobial β-Stranded [KL]n Peptides.

Amphipathic peptides can act as antibiotics due to membrane permeabilization. KL peptides with the repetitive sequence [Lys-Leu]n-NH2 form amphipathic β-strands in the presence of lipid bilayers. As they are known to kill bacteria in a peculiar length-dependent manner, we suggest here several different functional models, all of which seem plausible, including a carpet mechanism, a β-barrel pore, a toroidal wormhole, and a β-helix. To resolve their genuine mechanism, the activity of KL peptides with lengths from 6–26 amino acids (plus some inverted LK analogues) was systematically tested against bacteria and erythrocytes. Vesicle leakage assays served to correlate bilayer thickness and peptide length and to examine the role of membrane curvature and putative pore diameter. KL peptides with 10–12 amino acids showed the best therapeutic potential, i.e., high antimicrobial activity and low hemolytic side effects. Mechanistically, this particular window of an optimum β-strand length around 4 nm (11 amino acids × 3.7 Å) would match the typical thickness of a lipid bilayer, implying the formation of a transmembrane pore. Solid-state 15N- and 19F-NMR structure analysis, however, showed that the KL backbone lies flat on the membrane surface under all conditions. We can thus refute any of the pore models and conclude that the KL peptides rather disrupt membranes by a carpet mechanism. The intriguing length-dependent optimum in activity can be fully explained by two counteracting effects, i.e., membrane binding versus amyloid formation. Very short KL peptides are inactive, because they are unable to bind to the lipid bilayer as flexible β-strands, whereas very long peptides are inactive due to vigorous pre-aggregation into β-sheets in solution.

β subunits of GABAA receptors form proton-gated chloride channels: Insights into the molecular basis

Gamma-aminobutyric acid type A receptors (GABAARs) are ligand gated channels mediating inhibition in the central nervous system. Here, we identify a so far undescribed function of β-subunit homomers as proton-gated anion channels. Mutation of a single H267A in β3 subunits completely abolishes channel activation by protons. In molecular dynamic simulations of the β3 crystal structure protonation of H267 increased the formation of hydrogen bonds between H267 and E270 of the adjacent subunit leading to a pore stabilising ring formation and accumulation of Cl- within the transmembrane pore. Conversion of these residues in proton insensitive ρ1 subunits transfers proton-dependent gating, thus highlighting the role of this interaction in proton sensitivity. Activation of chloride and bicarbonate currents at physiological pH changes (pH50 is in the range 6- 6.3) and kinetic studies suggest a physiological role in neuronal and non-neuronal tissues that express beta subunits, and thus as potential novel drug target.

Cryo-EM elucidates mechanism of action of bacterial pore-forming toxins

Pore-forming toxins (PFTs) rupture plasma membranes and kill target cells. PFTs are secreted as soluble monomers that undergo drastic structural rearrangements upon interacting with the target membrane and generate transmembrane oligomeric pores. A detailed understanding of the molecular mechanisms of the pore-formation process remains unclear due to limited structural insights regarding the transmembrane oligomeric pore states of the PFTs. However, recent advances in the field of cryo-electron microscopy (cryo-EM) have led to the high-resolution structure determination of the oligomeric pore forms of diverse PFTs. Here, we discuss the pore-forming mechanisms of various PFTs, specifically the mechanistic details contributed by the cryo-EM-based structural studies.

Complete structure elucidation of a functional form of the Bacillus thuringiensis Cry4Ba δ-endotoxin: Insights into toxin-induced transmembrane pore architecture

The insecticidal nature of Cry δ-endotoxins produced by Bacillus thuringiensis is generally attributed to their ability to form transmembrane pores, causing lysis of target insect cells. Previously, the truncated tertiary structure of the chymotrypsin-treated Cry4Ba toxin lacking the N-terminal helices—α1 and α2 was reported. To elucidate a more complete functional structure, a 65-kDa trypsin-activated form of the Cry4Ba-R203Q mutant toxin was thus generated for X-ray crystallography by eliminating the Arg203-tryptic cleavage site. The 2.0 Å crystal structure of Cry4Ba-R203Q with R-factor of 21.5% and Rfree of 23.7.%, as subsequently improved with homology-based modeling and molecular dynamics (MD) simulations, revealed a wedge-shaped arrangement of three domains: a well-defined N-terminal domain of eight α-helices (α1, α2a, α2b, α3, α4, α5, α6 and α7) responsible for pore formation, a three-β-sheet prism displaying two functional motifs and a C-terminal β-sandwich domain. A full-atom structural model of the Cry4Ba pre-pore trimer constructed using a single-particle 3D-reconstructed template revealed that each toxin monomer forms the stable trimer by packing α3 and α4 together at the central interface. When MD simulations of a membrane-associated trimeric pore model comprising three α4-loop-α5 hairpins were performed, an stable open-pore structure at the membrane-water interface was clearly observed. Two conserved side-chains—Asn166 and Tyr170 in the α4-α5 loop were found to interact directly with phospholipid head groups, leading to pore opening and stability. Overall data provide the first complete view of the 3D structure of the Cry4Ba mosquito-active toxin and its trimeric pore architecture, underlining the importance of two critical loop residues—Asn166 and Tyr170.