Atomic force microscopy of membrane pore formation by cholesterol dependent cytolysins

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Bacteriocins: mechanism of membrane insertion and pore formation

Lactic acid bacteria produce several types of pore forming peptides. Class I bacteriocins are lantibiotics that contain (methyl)lanthionine residues that may form intramolecular thioether rings. These peptides generally have a broad spectrum of activity and form unstable pores. Class II bacteriocins are small, heat stable peptides mostly with a narrow spectrum of activity. Most bacteriocins interact with anionic lipids that are abundantly present in the membranes of Gram-positive bacteria.'Docking molecules' may enhance the conductivity and stability of lantibiotic pores, while'receptors' in the target membrane may determine specificity of class II bacteriocins. Insertion into the membrane of many bacteriocins is proton motive force driven. Lantibiotics may form pores according to a'wedge-like' model, while class II bacteriocins may enhance membrane permeability either by the formation of a'barrel stave' pore or by a'carpet' mechanism.     

Formation of ion-conducting channels by the membrane attack complex proteins of complement.

The effects of sequential additions of purified human complement proteins C5b-6, C7, C8, and C9 to assemble the C5b-9 membrane attack complex (MAC) of complement on electrical properties of planar lipid bilayers have been analyzed. The high resistance state of such membranes was impaired after assembly of large numbers of C5b-8 complexes as indicated by the appearance of rapidly fluctuating membrane currents. The C5b-8 induced conductance was voltage dependent and rectifying at higher voltages. Addition of C9 to membranes with very few C5b-8 complexes caused appearance of few discrete single channels of low conductance (5-25 pS) but after some time very large (greater than 0.5 nS) jumps in conductance could be monitored. This high macroscopic conductance state was dominated by 125-pS channels having a lifetime of approximately 1 s. The high conductance state was not stable and declined again after a period of 1-3 h. Incorporation of MAC extracted from complement-lysed erythrocytes into liposomes and subsequent transformation of such complexes into planar bilayers via an intermediate monolayer state resulted in channels with characteristics similar to the ones produced by sequential assembly of C5b-9. Comparison of the high-conductance C5b-9 channel characteristics (lifetime, ion preference, ionic-strength dependence) with those produced by poly(C9) (the circular or tubular aggregation product of C9) as published by Young, J.D.-E., Z.A. Cohn, and E.R. Podack. (1986. Science [Wash. DC]. 233:184-190.) indicates that the two are significantly different.     

PRELP protein inhibits the formation of the complement membrane attack complex

PRELP is a 58-kDa proteoglycan found in a variety of extracellular matrices, including cartilage and at several basement membranes. In rheumatoid arthritis (RA), the cartilage tissue is destroyed and fragmented molecules, including PRELP, are released into the synovial fluid where they may interact with components of the complement system. In a previous study, PRELP was found to interact with the complement inhibitor C4b-binding protein, which was suggested to locally down-regulate complement activation in joints during RA. Here we show that PRELP directly inhibits all pathways of complement by binding C9 and thereby prevents the formation of the membrane attack complex (MAC). PRELP does not interfere with the interaction between C9 and already formed C5b-8, but inhibits C9 polymerization thereby preventing formation of the lytic pore. The alternative pathway is moreover inhibited already at the level of C3-convertase formation due to an interaction between PRELP and C3. This suggests that PRELP may down-regulate complement attack at basement membranes and on damaged cartilage and therefore limit pathological complement activation in inflammatory disease such as RA. The net outcome of PRELP-mediated complement inhibition will highly depend on the local concentration of other complement modulating molecules as well as on the local concentration of available complement proteins.     

Insecticidal toxin complex proteins from Xenorhabdus nematophilus: structure and pore formation

Toxin complexes from Xenorhabdus and Photorhabdus spp. bacteria represent novel insecticidal proteins. We purified a native toxin complex (toxin complex 1) from Xenorhabdus nematophilus. The toxin complex is composed of three different proteins, XptA2, XptB1, and XptC1, representing products from class A, B, and C toxin complex genes, respectively. We showed that recombinant XptA2 and co-produced recombinant XptB1 and XptC1 bind together with a 4:1:1 stoichiometry. XptA2 forms a tetramer of ～1,120 kDa that bound to solubilized insect brush border membranes and induced pore formation in black lipid membranes. Co-expressed XptB1 and XptC1 form a tight 1:1 binary complex where XptC1 is C-terminally truncated, resulting in a 77-kDa protein. The ～30-kDa C-terminally cleaved portion of XptC1 apparently only loosely associates with this binary complex. XptA2 had only modest oral toxicity against lepidopteran insects but as a complex with co-produced XptB1 and XptC1 had high levels of insecticidal activity. Addition of co-expressed class B (TcdB2) and class C (TccC3) proteins from Photorhabdus luminescens to the Xenorhabdus XptA2 protein resulted in formation of a hybrid toxin complex protein with the same 4:1:1 stoichiometry as the native Xenorhabdus toxin complex 1. This hybrid toxin complex, like the native toxin complex, was highly active against insects.     

A novel mechanism of pore formation: membrane penetration by the N-terminal amphipathic region of equinatoxin

Equinatoxin II is a representative of actinoporins, eukaryotic pore-forming toxins from sea anemones. It creates pores in natural and artificial lipid membranes by an association of three or four monomers. Cysteine-scanning mutagenesis was used to study the structure of the N terminus, which is proposed to be crucial in transmembrane pore formation. We provide data for two steps of pore formation: a lipid-bound monomeric intermediate state and a final oligomeric pore. Results show that residues 10-28 are organized as an alpha-helix in both steps. In the first step, the whole region is transferred to a lipid-water interface, laying flat on the membrane. In the pore-forming state, the hydrophilic side of the amphipathic helix lines the pore lumen. The pore has a restriction around Asp-10, according to the permeabilization ratio of ions flowing through pores formed by chemically modified mutants. A general model was introduced to derive the tilt angle of the helix from the ion current data. This study reveals that actinoporins use a unique single helix insertion mechanism for pore formation.     

GSDMD membrane pore formation constitutes the mechanism of pyroptotic cell death

Pyroptosis is a lytic type of cell death that is initiated by inflammatory caspases. These caspases are activated within multi‐protein inflammasome complexes that assemble in response to pathogens and endogenous danger signals. Pyroptotic cell death has been proposed to proceed via the formation of a plasma membrane pore, but the underlying molecular mechanism has remained unclear. Recently, gasdermin D (GSDMD), a member of the ill‐characterized gasdermin protein family, was identified as a caspase substrate and an essential mediator of pyroptosis. GSDMD is thus a candidate for pyroptotic pore formation. Here, we characterize GSDMD function in live cells and in vitro. We show that the N‐terminal fragment of caspase‐1‐cleaved GSDMD rapidly targets the membrane fraction of macrophages and that it induces the formation of a plasma membrane pore. In vitro, the N‐terminal fragment of caspase‐1‐cleaved recombinant GSDMD tightly binds liposomes and forms large permeability pores. Visualization of liposome‐inserted GSDMD at nanometer resolution by cryo‐electron and atomic force microscopy shows circular pores with variable ring diameters around 20 nm. Overall, these data demonstrate that GSDMD is the direct and final executor of pyroptotic cell death.     

Outer membrane permeabilization by the membrane attack complex sensitizes Gram-negative bacteria to antimicrobial proteins in serum and phagocytes

Infections with Gram-negative bacteria form an increasing risk for human health due to antibiotic resistance. Our immune system contains various antimicrobial proteins that can degrade the bacterial cell envelope. However, many of these proteins do not function on Gram-negative bacteria, because the impermeable outer membrane of these bacteria prevents such components from reaching their targets. Here we show that complement-dependent formation of Membrane Attack Complex (MAC) pores permeabilizes this barrier, allowing antimicrobial proteins to cross the outer membrane and exert their antimicrobial function. Specifically, we demonstrate that MAC-dependent outer membrane damage enables human lysozyme to degrade the cell wall of E. coli. Using flow cytometry and confocal microscopy, we show that the combination of MAC pores and lysozyme triggers effective E. coli cell wall degradation in human serum, thereby altering the bacterial cell morphology from rod-shaped to spherical. Completely assembled MAC pores are required to sensitize E. coli to the antimicrobial actions of lysozyme and other immune factors, such as Human Group IIA-secreted Phospholipase A2. Next to these effects in a serum environment, we observed that the MAC also sensitizes E. coli to more efficient degradation and killing inside human neutrophils. Altogether, this study serves as a proof of principle on how different players of the human immune system can work together to degrade the complex cell envelope of Gram-negative bacteria. This knowledge may facilitate the development of new antimicrobials that could stimulate or work synergistically with the immune system.     

On the mechanism of pore formation by melittin

The mechanism of pore formation of lytic peptides, such as melittin from bee venom, is thought to involve binding to the membrane surface, followed by insertion at threshold levels of bound peptide. We show that in membranes composed of zwitterionic lipids, i.e. phosphatidylcholine, melittin not only forms pores but also inhibits pore formation. We propose that these two modes of action are the result of two competing reactions: direct insertion into the membrane and binding parallel to the membrane surface. The direct insertion of melittin leads to pore formation, whereas the parallel conformation is inactive and prevents other melittin molecules from inserting, hence preventing pore formation.     

Size-dependent leak of soluble and membrane proteins through the yeast nuclear pore complex

Nuclear pore complexes (NPCs) allow selective import and export while forming a barrier for untargeted proteins. Using fluorescence microscopy, we measured in vivo the permeability of the Saccharomyces cerevisiae NPC for multidomain proteins of different sizes and found that soluble proteins of 150 kDa and membrane proteins with an extralumenal domain of 90 kDa were still partly localized in the nucleus on a time scale of hours. The NPCs thus form only a weak barrier for the majority of yeast proteins, given their monomeric size. Using FGΔ-mutant strains, we showed that specific combinations of Nups, especially with Nup100, but not the total mass of FG-nups per pore, were important for forming the barrier. Models of the disordered phase of wild-type and mutant NPCs were generated using a one bead per amino acid molecular dynamics model. The permeability measurements correlated with the density predictions from coarse-grained molecular dynamics simulations in the center of the NPC. The combined in vivo and computational approach provides a framework for elucidating the structural and functional properties of the permeability barrier of nuclear pore complexes.     

Active metabolite of atorvastatin inhibits membrane cholesterol domain formation by an antioxidant mechanism

The advanced atherosclerotic lesion is characterized by the formation of microscopic cholesterol crystals that contribute to mechanisms of inflammation and apoptotic cell death. These crystals develop from membrane cholesterol domains, a process that is accelerated under conditions of hyperlipidemia and oxidative stress. In this study, the comparative effects of hydroxymethylglutaryl-CoA (HMG-CoA) reductase inhibitors (statins) on oxidative stress-induced cholesterol domain formation were tested in model membranes containing physiologic levels of cholesterol using small angle x-ray diffraction approaches. In the absence of HMG-CoA reductase, only the atorvastatin active o-hydroxy metabolite (ATM) blocked membrane cholesterol domain formation as a function of oxidative stress. This effect of ATM is attributed to electron donation and proton stabilization mechanisms associated with its phenoxy group located in the membrane hydrocarbon core. ATM inhibited lipid peroxidation in human low density lipoprotein and phospholipid vesicles in a dose-dependent manner, unlike its parent and other statins (pravastatin, rosuvastatin, simvastatin). These findings indicate an atheroprotective effect of ATM on membrane lipid organization through a potent antioxidant mechanism.     

Staphylococcal alpha-toxin: a study of membrane penetration and pore formation

Cell lysis by staphylococcal alpha-toxin, a potent virulence factor of most pathogenic strains of Staphylococcus aureus, follows a three-step sequence: binding of toxin to the membrane, leaking of ions caused by membrane injury, and rupturing of the membrane caused by osmotic swelling. The membrane injury step is composed of two separate events, membrane penetration and membrane perturbation. The membrane penetration event involves conversion of the soluble toxin monomer into an amphipathic molecule, which inserts into the lipid bilayer of the membrane. The membrane perturbation event involves association of the toxin monomers, in the plane of the membrane, to form hexameric transmembrane pores. In this study, we demonstrate that, in an asolectin liposome system, controlling the pH of the external buffer permits these two events to be temporally resolved. Using Controlled-Pore Glass bead-purified alpha-toxin, four events are measured as a function of pH: (a) release of potassium from prelabeled asolectin vesicles, (b) conversion of the toxin to a globally hydrophobic molecule, (c) binding of detergent by the toxin, and (d) labeling of the toxin with photoactivable, radiolabeled, hydrophobic probes. Two of these events, potassium release and conversion to a net hydrophobic state, are paired in that, for the event to occur, each requires a pH of 4.6 or less. In contrast, photolabeling with the membrane probes PC I and PC II (where PC represents phosphatidylcholine) is easily detectable at pH values as high as 5.0 and 6.0. These results demonstrate that, as the pH is lowered, two distinct changes in the physical properties of alpha-toxin occur. The first, which occurs under mild acidic conditions, converts the toxin from a water-soluble molecule into an amphipathic molecule. The second, requiring relatively more acidic conditions, converts the amphipathic toxin molecule into a globally hydrophobic molecule. Correlated with these physical changes in the alpha-toxin molecule is the acquisition of two new biological properties. The conversion of alpha-toxin into an amphipathic conformation correlates with the acquisition of the biological property of the reversible penetration into the bilayer of the asolectin liposome membrane, as evidenced by labeling with the photoactivable probes. At lower pH, the conversion of the toxin into a globally hydrophobic molecule correlates with the biological property of causing damage to the cell membrane, as measured by the release of internal potassium ions, presumably by the formation of transmembrane hexamer pores.(ABSTRACT TRUNCATED AT 400 WORDS)     

Recovery of human neutrophils from complement attack: removal of the membrane attack complex by endocytosis and exocytosis

Nucleated cells can resist lysis by and recover from complement attack even after formation of the potentially cytolytic membrane attack complex on the cell surface. We have found that human neutrophils resist complement lysis by the physical removal of membrane attack complexes by both endocytic and exocytic process. The latter mechanism predominates, vesiculation being detectable within 60 sec of initiating the complement cascade. Sixty-five percent of the formed complexes are removed on plasma membrane vesicles, although only 2% of the cell surface is lost. Ultrastructural examination revealed that these vesicles were covered with ring-like "classical" complement lesions. Analysis of these vesicles by gel electrophoresis indicated that C9 was present exclusively in the form of a sodium dodecyl sulfate-resistant, high m.w. complex. In contrast, the 35% of C9 that remained associated with the cells was found to be inaccessible to a C9-specific monoclonal antibody, and was partly degraded, suggesting internalization of the membrane attack complex and proteolysis of some C9 molecules. The molar ratio of C9 to C8 was 12 to 1 on shed vesicles and on recovered cells.     

The nucleoporin Nup188 controls passage of membrane proteins across the nuclear pore complex

All transport across the nuclear envelope (NE) is mediated by nuclear pore complexes (NPCs). Despite their enormous size, ∼60 MD in vertebrates, they are comprised of only ∼30 distinct proteins (nucleoporins or Nups), many of which form subcomplexes that act as building blocks for NPC assembly. One of these evolutionarily conserved subcomplexes, the Nup93 complex, is a major structural component linking the NPC to the membranes of the NE. Using in vitro nuclear assembly assays, we show that two components of the Nup93 complex, Nup188 and Nup205, are dispensable for NPC formation. However, nuclei lacking Nup188 increase in size by several fold compared with wild type. We demonstrate that this phenotype is caused by an accelerated translocation of integral membrane proteins through NPCs, suggesting that Nup188 confines the passage of membrane proteins and is thus crucial for the homeostasis of the different nuclear membranes.     

Lipid II-mediated pore formation by the peptide antibiotic nisin: a black lipid membrane study

The antibiotic peptide nisin is the first known lantibiotic that uses a docking molecule within the bacterial cytoplasmic membrane for pore formation. Through specific interaction with the cell wall precursor lipid II, nisin forms defined pores which are stable for seconds and have pore diameters of 2 to 2.5 nm.     

Biophysical and biochemical strategies to understand membrane binding and pore formation by sticholysins,pore-forming proteins from a sea anemone

Actinoporins constitute a unique class of pore-forming toxins found in sea anemones that are able to bind and oligomerize in membranes, leading to cell swelling, impairment of ionic gradients and, eventually, to cell death. In this review we summarize the knowledge generated from the combination of biochemical and biophysical approaches to the study of sticholysins I and II (Sts, StI/II), two actinoporins largely characterized by the Center of Protein Studies at the University of Havana during the last 20 years. These approaches include strategies for understanding the toxin structure–function relationship, the protein–membrane association process leading to pore formation and the interaction of toxin with cells. The rational combination of experimental and theoretical tools have allowed unraveling, at least partially, of the complex mechanisms involved in toxin–membrane interaction and of the molecular pathways triggered upon this interaction. The study of actinoporins is important not only to gain an understanding of their biological roles in anemone venom but also to investigate basic molecular mechanisms of protein insertion into membranes, protein–lipid interactions and the modulation of protein conformation by lipid binding. A deeper knowledge of the basic molecular mechanisms involved in Sts–cell interaction, as described in this review, will support the current investigations conducted by our group which focus on the design of immunotoxins against tumor cells and antigen-releasing systems to cell cytosol as Sts-based vaccine platforms.     

Modulation of erythrocyte membrane proteins by membrane cholesterol and lipid fluidity.

Human erythrocyte membranes were enriched or depleted of cholesterol and effects on membrane proteins assessed with a membrane-impermeant sulfhydryl reagent, [35S]glutathione-maleimide. Reaction of the probe with intact cells quantifies exofacial sulfhydryl groups and reaction with leaky ghost membranes permits quantification of endofacial sulfhydryl groups. The mean endofacial sulfhydryl titer of cholesterol-enriched membranes exceeded that of cholesterol-depleted membrane by approximately 45 nmol/mg of protein or 64%. The corresponding exofacial titer of cholesterol-enriched cells was less than that of cholesterol-depleted cells by approximately 0.4 nmol/mg of protein, or 14%. Labeled membranes were examined by autoradiography of sodium dodecyl sulfate-polyacrylamide gel electropherograms to determine the labeling patterns of individual protein bands. Cholesterol enrichment enhanced the surface labeling of Coomassie brilliant blue stained bands 1,2,3, and 5, decreased the labeling of band 6, and did not change significantly that of band 4. The results demonstrate that changes in membrane cholesterol which influence lipid fluidity can alter the surface labeling of both intrinsic and extrinsic membrane proteins.     

Mapping the targeted membrane pore formation mechanism by solution NMR: the nisin Z and lipid II interaction in SDS micelles

Nisin is an example of type-A lantibiotics that contain cyclic lanthionine rings and unusual dehydrated amino acids. Among the numerous pore-forming antimicrobial peptides, type-A lantibiotics form an unique family of post-translationally modified peptides. Via the recognition of cell wall precursor lipid II, nisin has the capacity to form pores against Gram-positive bacteria with an extremely high activity in the nanomolar (nM) range. Here we report a high-resolution NMR spectroscopy study of nisin/lipid II interactions in SDS micelles as a model membrane system in order to elucidate the mechanism of molecular recognition at residue level. The binding to lipid II was studied through (15)N-(1)H HSQC titration, backbone amide proton temperature coefficient analysis, and heteronuclear (15)N[(1)H]-NOE relaxation dynamics experiments. Upon the addition of lipid II, significant changes were monitored in the N-terminal part of nisin. An extremely low amide proton temperature coefficient (Delta delta/Delta T) was found for the amide proton of Ala3 (> -0.1 ppb/K) in the complex form. This suggests tight hydrogen bonding and/or isolation from the bulk solvent for this residue. Large chemical shift perturbations were also observed in the first two rings. In contrast, the C-terminal part of nisin was almost unaffected. This part of the molecule remains flexible and solvent-exposed. On the basis of our results, a multistep pore-forming mechanism is proposed. The N-terminal part of nisin first binds to lipid II, and a subsequent structural rearrangement takes place. The C-terminal part of nisin is possibly responsible for the activation of the pore formation. In light of the emerging antibiotic resistance problems, an understanding of the specific recognition mechanism of nisin with lipid II at the residue specific level may therefore aid in the development of novel antibiotics.