BCL-2-family protein tBID can act as a BAX-like effector of apoptosis.

During apoptosis, the BCL-2-family protein tBID promotes mitochondrial permeabilization by activating BAX and BAK and by blocking anti-apoptotic BCL-2 members. Here, we report that tBID can also mediate mitochondrial permeabilization by itself, resulting in release of cytochrome c and mitochondrial DNA, caspase activation and apoptosis even in absence of BAX and BAK. This previously unrecognized activity of tBID depends on helix 6, homologous to the pore-forming regions of BAX and BAK, and can be blocked by pro-survival BCL-2 proteins. Importantly, tBID-mediated mitochondrial permeabilization independent of BAX and BAK is physiologically relevant for SMAC release in the immune response against Shigella infection. Furthermore, it can be exploited to kill leukaemia cells with acquired venetoclax resistance due to lack of active BAX and BAK. Our findings define tBID as an effector of mitochondrial permeabilization in apoptosis and provide a new paradigm for BCL-2 proteins, with implications for anti-bacterial immunity and cancer therapy.

Estimates of differential toxin expression governing heterogeneous intracellular lifespans of Streptococcus pneumoniae.

Following invasion of the host cell, pore-forming toxins secreted by pathogens compromise vacuole integrity and expose the microbe to diverse intracellular defence mechanisms. However, the quantitative correlation between toxin expression levels and consequent pore dynamics, fostering the intracellular life of pathogens, remains largely unexplored. In this study, using Streptococcus pneumoniae and its secreted pore-forming toxin pneumolysin (Ply) as a model system, we explored various facets of host-pathogen interactions in the host cytosol. Using time-lapse fluorescence imaging, we monitored pore formation dynamics and lifespans of different pneumococcal subpopulations inside host cells. Based on experimental histograms of various event timescales such as pore formation time, vacuolar death or cytosolic escape time and total degradation time, we developed a mathematical model based on first-passage processes that could correlate the event timescales to intravacuolar toxin accumulation. This allowed us to estimate Ply production rate, burst size and threshold Ply quantities that trigger these outcomes. Collectively, we present a general method that illustrates a correlation between toxin expression levels and pore dynamics, dictating intracellular lifespans of pathogens.

Activation energy for pore opening in lipid membranes under an electric field.

The standard model of pore formation was introduced more than fifty years ago, and it has been since, despite some refinements, the cornerstone for interpreting experiments related to pores in membranes. A central prediction of the model concerning pore opening under an electric field is that the activation barrier for pore formation is lowered proportionally to the square of the electric potential. However, this has only been scarcely and inconclusively confronted to experiments. In this paper, we study the electropermeability of model lipid membranes composed of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) containing different fractions of POPC-OOH, the hydroperoxidized form of POPC, in the range 0 to 100 mol %. By measuring ion currents across a 50-µm-diameter black lipid membrane (BLM) with picoampere and millisecond resolution, we detect hydroperoxidation-induced changes to the intrinsic bilayer electropermeability and to the probability of opening angstrom-size or larger pores. Our results over the full range of lipid compositions show that the energy barrier to pore formation is lowered linearly by the absolute value of the electric field, in contradiction with the predictions of the standard model.

An amphipathic Bax core dimer forms part of the apoptotic pore wall in the mitochondrial␣membrane.

Bax proteins form pores in the mitochondrial outer membrane to initiate apoptosis. This might involve their embedding in the cytosolic leaflet of the lipid bilayer, thus generating tension to induce a lipid pore with radially arranged lipids forming the wall. Alternatively, Bax proteins might comprise part of the pore wall. However, there is no unambiguous structural evidence for either hypothesis. Using NMR, we determined a high-resolution structure of the Bax core region, revealing a dimer with the nonpolar surface covering the lipid bilayer edge and the polar surface exposed to water. The dimer tilts from the bilayer normal, not only maximizing nonpolar interactions with lipid tails but also creating polar interactions between charged residues and lipid heads. Structure-guided mutations demonstrate the importance of both types of protein-lipid interactions in Bax pore assembly and core dimer configuration. Therefore, the Bax core dimer forms part of the proteolipid pore wall to permeabilize mitochondria.

Effect of membrane tension on antimicrobial peptide PGLa-induced pore formation in lipid bilayers.

The osmotic pressure (Π) method has recently been developed to quantitatively examine the effect of membrane tension (σ) on pore formation in giant unilamellar vesicles (GUVs) induced by antimicrobial peptides (AMPs). Here, we used the Π method to reveal the effect of σ on the interaction of an AMP, PGLa, with lipid bilayers comprising dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC) (4/6). PGLa induced leakage of fluorescent probes from single GUVs under Π, indicating nanopore formation. Membrane tension did not transform a PGLa-induced nanopore into a micropore nor cause GUV burst up to 3.4 mN/m, which is in contrast with the effect of σ on another AMP, magainin 2-induced pore formation, where lower σ resulted in GUV burst. The fraction of leaking GUVs at a specific time increased with increasing σ, indicating that the rate of PGLa-induced pore formation increases with increasing σ. The rate of transfer of fluorescent probe-labeled PGLa across the lipid bilayer without pore formation also increased with increasing σ. PGLa-induced pore formation requires a symmetric distribution of peptides in both leaflets of the GUV bilayer, and thus we infer that the increase in the rate of PGLa transfer from the outer leaflet to the inner leaflet underlies the increase in the rate of pore formation with increasing σ. On the basis of these results, we discuss the difference between the effect of σ on nanopore formation in GUV membranes induced by PGLa and that by magainin 2.

Conformational Switch of a Peptide Provides a Novel Strategy to Design Peptide Loaded Porous Organic Polymer for Pyroptosis Pathway Mediated Cancer Therapy.

While peptide-based drug development is extensively explored, this strategy has limitations due to rapid excretion from the body (or shorter half-life in the body) and vulnerability to protease-mediated degradation. To overcome these limitations, a novel strategy for the development of a peptide-based anticancer agent is introduced, utilizing the conformation switch property of a chameleon sequence stretch (PEP1) derived from a mycobacterium secretory protein, MPT63. The selected peptide is then loaded into a new porous organic polymer (PG-DFC-POP) synthesized using phloroglucinol and a cresol derivative via a condensation reaction to deliver the peptide selectively to cancer cells. Utilizing ensemble and single-molecule approaches, this peptide undergoes a transition from a disordered to an alpha-helical conformation, triggered by the acidic environment within cancer cells that is demonstrated. This adopted alpha-helical conformation resulted in the formation of proteolysis-resistant oligomers, which showed efficient membrane pore-forming activity selectively for negatively charged phospholipids accumulated in cancer cell membranes. The experimental results demonstrated that the peptide-loaded PG-DFC-POP-PEP1 exhibited significant cytotoxicity in cancer cells, leading to cell death through the Pyroptosis pathway, which is established by monitoring numerous associated events starting from lysosome membrane damage to GSDMD-induced cell membrane demolition. This novel conformational switch-based drug design strategy is believed to have great potential in endogenous environment-responsive cancer therapy and the development of future drug candidates to mitigate cancers.

COBL7 is required for stomatal formation via regulation of cellulose deposition in Arabidopsis.

As a key regulator of plant photosynthesis, water use efficiency and immunity, stomata are specialized cellular structures that adopt defined shapes. However, our knowledge about the genetic players of stomatal pore formation and stomatal morphogenesis remains limited. Forward genetic screening, positional cloning, confocal and electron microscopy, physiological and pharmacological assays were employed for isolation and characterization of mutants and genes. We identified a mutant, dsm1, with impaired cytokinesis and deformed stomata. DSM1 is highly expressed in guard mother cells and guard cells, and encodes COBRA-LIKE 7 (COBL7), a plant-specific glycosylphosphatidylinositol (GPI)-anchored protein. COBRA-LIKE 7 and its closest homologue, COBL8, are first enriched on the forming cell plates during cytokinesis, and then their subcellular distribution and abundance change are correlated with the progressive stages of stomatal pore formation. Both COBL7 and COBL8 possess an ability to bind cellulose. Perturbing the expression of COBL7 and COBL8 leads to a decrease in cellulose content and inhibition of stomatal pore development. Moreover, we found that COBL7, COBL8 and CSLD5 have synergistic effects on stomatal development and plant growth. Our findings reveal that COBL7 plays a predominant and functionally redundant role with COBL8 in stomatal formation through regulating cellulose deposition and ventral wall modification in Arabidopsis.

Synthetic antimicrobial peptides: activity against vancomycin-resistant Enterococcus faecalis and modulation of chloramphenicol antibacterial activity.

AIMS: Enterococcus faecalis (E. faecalis) is a leading cause of nosocomial infection and presents a wide spectrum of antibiotic resistance, being vancomycin-resistant Enterococcus (VRE) one of the most relevant. Synthetic antimicrobial peptides (SAMPs) are currently a promising option to overcome antimicrobial resistance. Thus, the purpose of this study was to assess the effect of eight SAMPs against vancomycin-resistant E. faecalis, as well as to investigate their mechanism of action and synergy with conventional antibiotics. METHODS AND RESULTS: Here, eight SAMPs, Mo-CBP3-PepI, Mo-CBP3-PepII, Mo-CBP3-PepIII, RcAlb-PepI, RcAlb-PepII, RcAlb-PepIII, PepGAT, and PepKAA, were tested for antibacterial activity in vitro against E. faecalis (ATCC® 51299) through broth microdilution. A maximum of 48% of E. faecalis growth inhibition was achieved by treatment with SAMPs alone. However, when these peptides were combined with the antibiotic chloramphenicol, assessed by checkerboard method, the inhibition increased to 55%-76% of inhibition, two to three-folds of increase if compared to the effects of the compounds alone. Microscopic analysis showed that E. faecalis cells treated with a combination of SAMPs and chloramphenicol resulted in bacterial membrane damage. The biofilm inhibition maximum was 22% for SAMPs alone, when combined with chloramphenicol, the maximum increased to 33%. CONCLUSIONS: SAMPs and their combination with chloramphenicol demonstrate antibacterial activity against E. faecalis, possibly by inducing bacterial membrane damage.

Exploring conventional and emerging dehydration technologies for slurry/liquid food matrices and their impact on porosity of powders: A comprehensive review.

The contribution of dehydration to the growing market of food powders from slurry/liquid matrices is inevitable. To overcome the challenges posed by conventional drying technologies, several innovative approaches have emerged. However, industrial implementation is limited due to insufficient information on the best-suited drying technologies for targeted products. Therefore, this review aimed to compare various conventional and emerging dehydration technologies (such as active freeze, supercritical, agitated thin-film, and vortex chamber drying) based on their fundamental principles, potential applications, and limitations. Additionally, this article reviewed the effects of drying technologies on porosity, which greatly influence the solubility, rehydration, and stability of powder. The comparison between different drying technologies enables informed decision-making in selecting the appropriate one. It was found that active freeze drying is effective in producing free-flowing powders, unlike conventional freeze drying. Vortex chamber drying could be considered a viable alternative to spray drying, requiring a compact chamber than the large tower needed for spray drying. Freeze-dried, spray freeze-dried, and foam mat-dried powders exhibit higher porosity than spray-dried ones, whereas supercritical drying produces nano-porous interconnected powders. Notably, several factors like glass transition temperature, drying technologies, particle aggregation, agglomeration, and sintering impact powder porosity. However, some binders, such as maltodextrin, sucrose, and lactose, could be applied in controlled agglomeration to enhance powder porosity. Further investigation on the effect of emerging technologies on powder properties and their commercial feasibility is required to discover their potential in liquid drying. Moreover, utilizing clean-label drying ingredients like dietary fibers, derived from agricultural waste, presents promising opportunities.

The estimation of pore size distribution of electroporated MCF-7 cell membrane.

The size of the pores created by external electrical pulses is important for molecule delivery into the cell. The size of pores and their distribution on the cell membrane determine the efficiency of molecule transport into the cell. There are very few studies visualizing the presence of electropores. In this study, we aimed to investigate the size distribution of electropores that were created by high intensity and short duration electrical pulses on MCF-7 cell membrane. Scanning Electron Microscopy (SEM) was used to visualize and characterize the membrane pores created by the external electric field. Structural changes on the surface of the electroporated cell membrane was observed by Atomic Force Microscopy (AFM). The size distribution of pore sizes was obtained by measuring the radius of 500 electropores. SEM imaging showed non-uniform patterning. The average radius of the electropores was 12 nm, 51.60% of pores were distributed within the range of 5 to 10 nm, and 81% of pores had radius below 15 nm. These results showed that microsecond (µs) high intensity electrical pulses cause the creation of heterogeneous nanopores on the cell membrane.

Electroporation is a phenomenon in which permeability of the cell membrane to molecules and ions is increased due to externally applied high electric field pulses. The externally applied electric field pulses create pores on the cell membrane, allowing ions and molecules that normally can not pass through the membrane. The transport of molecules into the cell is related to the size and distribution of the pores created on the membrane. Studies visualizing the presence of electropores are very limited. In this study, we aimed to visualize pores and determine the size distribution of pores created due to the application of external electric field pulses on the cell membrane of human breast cancer cells. The membrane pores created by external electric field were visualized and characterized by different imaging techniques. The size distribution of pores was obtained by measuring the radius of 500 pores created on the cell membrane due to the applied electric fields. The surface of the electropermeabilized cells were very rough due to deformation during electroporation. We observed heterogeneous pore populations that were formed due to application of external electrical pulses on the surface of cell membrane. The average radius of the pores was found to be 12 nm.

R-Type Fonticins Produced by Pragia fontium Form Large Pores with High Conductance.

Fonticins are phage tail-like bacteriocins produced by the Gram-negative bacterium Pragia fontium from the family Budviciaceae. This bacterium produces contractile-type particles that adsorb on the surface of sensitive bacteria and penetrate the cell wall, probably during contraction, in a way similar to the type VI secretion system. We characterized the pore-forming activity of fonticins using both living cells and in vitro model membranes. Using a potassium leakage assay, we show that fonticins are able to permeabilize sensitive cells. On black lipid membranes, single-pore conductance is about 0.78 nS in 1 M NaCl and appears to be linearly dependent on the increasing molar strength of NaCl solution, which is a property of considerably large pores. In agreement with these findings, fonticins are not ion selective for Na+, K+, and Cl-. Polyethylene glycol 3350 (PEG 3350) molecules of about 3.5 nm in diameter can enter the fonticin pore lumen, whereas the larger molecules cannot pass the pore. The size of fonticin pores was confirmed by transmission electron microscopy. The terminal membrane-piercing complex of the fonticin tube probably creates a selective barrier restricting passage of macromolecules. IMPORTANCE Phage tail-like bacteriocins are now the subject of research as potent antibacterial agents due to their narrow host specificity and single-hit mode of action. In this work, we focused on the structure and mode of action of fonticins. According to some theories, related particles were initially adapted for passage of double-stranded DNA (dsDNA) molecules, but fonticins changed their function during the evolution; they are able to form large pores through the bacterial envelope of Gram-negative bacteria. As various pore-forming proteins are extensively used for nanopore sequencing and stochastic sensing, we decided to investigate the pore-forming properties of fonticin protein complexes on artificial lipid membranes. Our research revealed remarkable structural properties of these particles that may have a potential application as a nanodevice.

Implications of conformational flexibility, lipid binding, and regulatory domains in cell-traversal protein CelTOS for apicomplexan migration.

Malaria and other apicomplexan-caused diseases affect millions of humans, agricultural animals, and pets. Cell traversal is a common feature used by multiple apicomplexan parasites to migrate through host cells and can be exploited to develop therapeutics against these deadly parasites. Here, we provide insights into the mechanism of the Cell-traversal protein for ookinetes and sporozoites (CelTOS), a conserved cell-traversal protein in apicomplexan parasites and malaria vaccine candidate. CelTOS has previously been shown to form pores in cell membranes to enable traversal of parasites through cells. We establish roles for the distinct protein regions of Plasmodium vivax CelTOS and examine the mechanism of pore formation. We further demonstrate that CelTOS dimer dissociation is required for pore formation, as disulfide bridging between monomers inhibits pore formation, and this inhibition is rescued by disulfide-bridge reduction. We also show that a helix-destabilizing amino acid, Pro127, allows CelTOS to undergo significant conformational changes to assemble into pores. The flexible C terminus of CelTOS is a negative regulator that limits pore formation. Finally, we highlight that lipid binding is a prerequisite for pore assembly as mutation of a phospholipids-binding site in CelTOS resulted in loss of lipid binding and abrogated pore formation. These findings identify critical regions in CelTOS and will aid in understanding the egress mechanism of malaria and other apicomplexan parasites as well as have implications for studying the function of other essential pore-forming proteins.

Membrane binding and pore forming insertion of PEX5 into horizontal lipid bilayer.

The assembly of the peroxisomal translocon involves the transition of a soluble form of the peroxisomal targeting receptor PEX5 into a membrane-bound form, which becomes an integral membrane component of the import pore for peroxisomal matrix proteins. How this transition occurs is still a mystery. We addressed this question using a artificial horizontal bilayer in combination with fluorescence time-correlated single photon counting (TCSPC) and electrophysiological channel recording. Purified human isoform PEX5L and truncated PEX5L(1-335) lacking the cargo binding domain were selectively labeled with thiol-reactive Atto-dyes. Diffusion coefficients of labeled protein in solution show that PEX5L is monomeric with a rather compact spherical conformation, while the truncated protein appeared in a more extended conformation. Labeled PEX5L and the truncated PEX5L(1-335) bind stably to horizontal bilayer thereby accumulating around 100-fold. The diffusion coefficients of the membrane-bound PEX5L forms are 3-4 times lower than in solution, indicating the formation of larger complexes. Electrophysiological single channel recording shows that membrane-bound labeled and non-labeled PEX5L, but not the truncated PEX5L(1-335), can form ion conducting membrane channels. The data suggest that PEX5L is the pore-forming component of the oligomeric peroxisomal translocon and that spontaneous PEX5L membrane surface binding might be an important step in its assembly.

Visualizing the Domino-Like Prepore-to-Pore Transition of Streptolysin O by High-Speed AFM.

Pore-forming proteins (PFPs) are produced by various organisms, including pathogenic bacteria, and form pores within the target cell membrane. Streptolysin O (SLO) is a PFP produced by Streptococcus pyogenes and forms high-order oligomers on the membrane surface. In this prepore state, multiple α-helices in domain 3 of each subunit exist as unfolded structures and transiently interact with each other. They subsequently transition into transmembrane ß-hairpins (TMHs) and form pores with diameters of 20-30 nm. However, in this pore formation process, the trigger of the transition in a subunit and collaboration between subunits remains elusive. Here, I observed the dynamic pore formation process using high-speed atomic force microscopy. During the oligomer transition process, each subunit was sequentially inserted into the membrane, propagating along the oligomer in a domino-like fashion (chain reaction). This process also occurred on hybrid oligomers containing wildtype and mutant subunits, which cannot insert into the membrane because of an introduced disulfide bond. Furthermore, propagation still occurred when an excessive force was added to hybrid oligomers in the prepore state. Based on the observed chain reactions, I estimate the free energies and forces that trigger the transition in a subunit. Furthermore, I hypothesize that the collaboration between subunits is related to the structure of their TMH regions and interactions between TMH-TMH and TMH-lipid molecules.

The Functionality of Membrane-Inserting Proteins and Peptides: Curvature Sensing, Generation, and Pore Formation.

Proteins and peptides with hydrophobic and amphiphilic segments are responsible for many biological functions. The sensing and generation of membrane curvature are the functions of several protein domains or motifs. While some specific membrane proteins play an essential role in controlling the curvature of distinct intracellular membranes, others participate in various cellular processes such as clathrin-mediated endocytosis, where several proteins sort themselves at the neck of the membrane bud. A few membrane-inserting proteins form nanopores that permeate selective ions and water to cross the membrane. In addition, many natural and synthetic small peptides and protein toxins disrupt the membrane by inducing nonspecific pores in the membrane. The pore formation causes cell death through the uncontrolled exchange between interior and exterior cellular contents. In this article, we discuss the insertion depth and orientation of protein/peptide helices, and their role as a sensor and inducer of membrane curvature as well as a pore former in the membrane. We anticipate that this extensive review will assist biophysicists to gain insight into curvature sensing, generation, and pore formation by membrane insertion.

Membrane Interactions and Uncoating of Aichi Virus, a Picornavirus That Lacks a VP4.

Kobuviruses are an unusual and poorly characterized genus within the picornavirus family and can cause gastrointestinal enteric disease in humans, livestock, and pets. The human kobuvirus Aichi virus (AiV) can cause severe gastroenteritis and deaths in children below the age of 5 years; however, this is a very rare occurrence. During the assembly of most picornaviruses (e.g., poliovirus, rhinovirus, and foot-and-mouth disease virus), the capsid precursor protein VP0 is cleaved into VP4 and VP2. However, kobuviruses retain an uncleaved VP0. From studies with other picornaviruses, it is known that VP4 performs the essential function of pore formation in membranes, which facilitates transfer of the viral genome across the endosomal membrane and into the cytoplasm for replication. Here, we employ genome exposure and membrane interaction assays to demonstrate that pH plays a critical role in AiV uncoating and membrane interactions. We demonstrate that incubation at low pH alters the exposure of hydrophobic residues within the capsid, enhances genome exposure, and enhances permeabilization of model membranes. Furthermore, using peptides we demonstrate that the N terminus of VP0 mediates membrane pore formation in model membranes, indicating that this plays an analogous function to VP4. IMPORTANCE To initiate infection, viruses must enter a host cell and deliver their genome into the appropriate location. The picornavirus family of small nonenveloped RNA viruses includes significant human and animal pathogens and is also a model to understand the process of cell entry. Most picornavirus capsids contain the internal protein VP4, generated from cleavage of a VP0 precursor. During entry, VP4 is released from the capsid. In enteroviruses this forms a membrane pore, which facilitates genome release into the cytoplasm. Due to high levels of sequence similarity, it is expected to play the same role for other picornaviruses. Some picornaviruses, such as Aichi virus, retain an intact VP0, and it is unknown how these viruses rearrange their capsids and induce membrane permeability in the absence of VP4. Here, we have used Aichi virus as a model VP0 virus to test for conservation of function between VP0 and VP4. This could enhance understanding of pore function and lead to development of novel therapeutic agents that block entry.

The interactions of amyloid ß aggregates with phospholipid membranes and the implications for neurodegeneration.

Misfolding, aggregation and accumulation of Amyloid-ß peptides (Aß) in neuronal tissue and extracellular matrix are hallmark features of Alzheimer's disease (AD) pathology. Soluble Aß oligomers are involved in neuronal toxicity by interacting with the lipid membrane, compromising its integrity, and affecting the function of receptors. These facts indicate that the interaction between Aß oligomers and cell membranes may be one of the central molecular level factors responsible for the onset of neurodegeneration. The present review provides a structural understanding of Aß neurotoxicity via membrane interactions and contributes to understanding early events in Alzheimer's disease.

Disruption of plant plasma membrane by Nep1-like proteins in pathogen-plant interactions.

Lipid membrane destruction by microbial pore-forming toxins (PFTs) is a ubiquitous mechanism of damage to animal cells, but is less prominent in plants. Nep1-like proteins (NLPs) secreted by phytopathogens that cause devastating crop diseases, such as potato late blight, represent the only family of microbial PFTs that effectively damage plant cells by disrupting the integrity of the plant plasma membrane. Recent research has elucidated the molecular mechanism of NLP-mediated membrane damage, which is unique among microbial PFTs and highly adapted to the plant membrane environment. In this review, we cover recent insight into how NLP cytolysins damage plant membranes and cause cell death.

Synthetic amino acids-derived peptides target Cryptococcus neoformans by inducing cell membrane disruption.

We investigated synthetic amino acid-based approach to design short peptide-based antibiotics. Tautomerically restricted, amphiphilic 1-aryl-l-histidines along with hydrophobic tryptophan were utilized to synthesize the designed peptides. l-Trp-l-His(1-biphenyl)-NHBzl (12e, IC50 = 1.91 µg/mL; MIC = 3.46 µg/mL) and l-His[1-(4-n-butylphenyl)]-l-Trp-l-His[1-(4-n-butylphenyl)]-NHBzl (16d, IC50 = 1.36 µg/mL; MIC = 2.46 µg/mL) produced potency against Cryptococcus neoformans. Peptides with moderate antibacterial activities (IC50s = 4.40-8.80 µg/mL) were also identified. The mechanism of action and cellular changes revealed that membrane disruption due to interactions of the positively charged peptides with the negatively charged membrane of the cryptococcal cells result in permeabilization, leading to pore formation. The internal localization of the peptides instigated the interactions with DNA causing fragmentation of the genetic material, which together with membrane disruption led to cell death. Flow cytometric analysis points to cells death by apoptotic pathway. Time kill kinetics and synergistic study confirmed the fungicidal nature and synergism with amphotericin B.

Channel Formation in Cry Toxins: An Alphafold-2 Perspective.

Bacillus thuringiensis (Bt) strains produce pore-forming toxins (PFTs) that attack insect pests. Information for pre-pore and pore structures of some of these Bt toxins is available. However, for the three-domain (I-III) crystal (Cry) toxins, the most used Bt toxins in pest control, this crucial information is still missing. In these Cry toxins, biochemical data have shown that 7-helix domain I is involved in insertion in membranes, oligomerization and formation of a channel lined mainly by helix α4, whereas helices α1 to α3 seem to have a dynamic role during insertion. In the case of Cry1Aa, toxic against Manduca sexta larvae, a tetrameric oligomer seems to precede membrane insertion. Given the experimental difficulty in the elucidation of the membrane insertion steps, we used Alphafold-2 (AF2) to shed light on possible oligomeric structural intermediates in the membrane insertion of this toxin. AF2 very accurately (<1 Å RMSD) predicted the crystal monomeric and trimeric structures of Cry1Aa and Cry4Ba. The prediction of a tetramer of Cry1Aa, but not Cry4Ba, produced an 'extended model' where domain I helices α3 and α2b form a continuous helix and where hydrophobic helices α1 and α2 cluster at the tip of the bundle. We hypothesize that this represents an intermediate that binds the membrane and precedes α4/α5 hairpin insertion, together with helices α6 and α7. Another Cry1Aa tetrameric model was predicted after deleting helices α1 to α3, where domain I produced a central cavity consistent with an ion channel, lined by polar and charged residues in helix α4. We propose that this second model corresponds to the 'membrane-inserted' structure. AF2 also predicted larger α4/α5 hairpin n-mers (14 ≤n ≤ 17) with high confidence, which formed even larger (~5 nm) pores. The plausibility of these models is discussed in the context of available experimental data and current paradigms.