EPR and FTIR studies reveal the importance of highly ordered sterol-enriched membrane domains for ostreolysin activity.

Ostreolysin is a cytolytic protein from the edible oyster mushroom (Pleurotus ostreatus), which recognizes specifically and binds to raft-like sterol-enriched membrane domains that exist in the liquid-ordered phase. Its binding can be abolished by micromolar concentrations of lysophospholipids and fatty acids. The membrane activity of ostreolysin, however, does not completely correlate with the ability of a certain sterol to induce the formation of a liquid-ordered phase, suggesting that the protein requires an additional structural organization of the membrane to exert its activity. The aim of this study was to further characterize the lipid membranes that facilitate ostreolysin binding by analyzing their lipid phase domain structure. Fourier-transformed infrared spectroscopy (FTIR) and electron paramagnetic resonance (EPR) were used to analyze the ordering and dynamics of membrane lipids and the membrane domain structure of a series of unilamellar liposomes prepared by systematically changing the lipid components and their ratios. Our results corroborate the earlier conclusion that the average membrane fluidity of ostreolysin-susceptible liposomes alone cannot account for the membrane activity of the protein. Combined with previous data computer-aided interpretation of EPR spectra strongly suggests that chemical properties of membrane constituents, their specific distribution, and physical characteristics of membrane nanodomains, resulting from the presence of sterol and sphingomyelin (or a highly ordered phospholipid, dipalmitoylphosphatidylcholine), are essential prerequisites for ostreolysin membrane binding and pore-formation.

Structural features of distinctin affecting peptide biological and biochemical properties.

The antimicrobial peptide distinctin consists of two peptide chains linked by a disulfide bridge; it presents a peculiar fold in water resulting from noncovalent dimerization of two heterodimeric molecules. To investigate the contribution of each peptide chain and the S-S bond to distinctin biochemical properties, different monomeric and homodimeric peptide analogues were synthesized and comparatively evaluated with respect to the native molecule. Our experiments demonstrate that the simultaneous occurrence of both peptide chains and the disulfide bond is essential for the formation of the quaternary structure of distinctin in aqueous media, able to resist protease action. In contrast, distinctin and monomeric and homodimeric analogues exhibited comparable antimicrobial activities, suggesting only a partial contribution of the S-S bond to peptide killing effectiveness. Relative bactericidal properties paralleled liposome permeabilization results, definitively demonstrating that microbial membranes are the main target of distinctin activity. Various biophysical experiments performed in membrane-mimicking media, before and after peptide addition, provided information about peptide secondary structure, lipid bilayer organization, and lipid-peptide orientation with respect to membrane surface. These data were instrumental in the generation of putative models of peptide-lipid supramolecular pore complexes.

Biological characterization of white line-inducing principle (WLIP) produced by Pseudomonas reactans NCPPB1311.

The biological activities of the lipodepsipeptides (LDP) white line-inducing principle (WLIP), produced by Pseudomonas reactans NCPPB1311, and tolaasin I, produced by R tolaasii NCPPB2192, were compared. Antimicrobial assays showed that both LDP inhibited the growth of fungi-including the cultivated mushrooms Agaricus bisporus, Lentinus edodes, and Pleurotus spp.--chromista, and gram-positive bacteria. Assays of the two LDP on blocks of Agaricus bisporus showed their capacity to alter the mushrooms' pseudo-tissues though WLIP was less active than that of tolaasin I. Contrary to previous studies, tolaasin I was found to inhibit the growth of gram-negative bacteria belonging to the genera Escherichia, Erwinia, Agrobacterium, Pseudomonas, and Xanthomonas. The only gram-negative bacterium affected by WLIP was Erwinia carotovora subsp. carotovora. Both WLIP and tolaasin I caused red blood cell lysis through a colloid-osmotic shock mediated by transmembrane pores; however, the haemolytic activity of WLIP was greater than that of tolaasin I. Transmembrane pores, at a concentration corresponding to 1.5 x C50, showed a radius between 1.5 and 1.7 +/- 0.1 nm for WLIP and 2.1 +/- 0.1 nm for tolaasin I. The antifungal activity of WLIP together with the finding that avirulent morphological variants of P. reactans lack WLIP production suggests that WLIP may play an important role in the interaction of the producing bacterium P. reactans and cultivated mushrooms.

Peptides corresponding to helices 5 and 6 of Bax can independently form large lipid pores.

Proteins of the B-cell lymphoma protein 2 (Bcl2) family are key regulators of the apoptotic cascade, controlling the release of apoptotic factors from the mitochondrial intermembrane space. A helical hairpin found in the core of water-soluble folds of these proteins has been reported to be the pore-forming domain. Here we show that peptides including any of the two alpha-helix fragments of the hairpin of Bcl2 associated protein X (Bax) can independently induce release of large labelled dextrans from synthetic lipid vesicles. The permeability promoted by these peptides is influenced by intrinsic monolayer curvature and accompanied by fast transbilayer redistribution of lipids, supporting a toroidal pore mechanism as in the case of the full-length protein. However, compared with the pores made by complete Bax, the pores made by the Bax peptides are smaller and do not need the concerted action of tBid. These data indicate that the sequences of both fragments of the hairpin contain the principal physicochemical requirements for pore formation, showing a parallel between the permeabilization mechanism of a complex regulated protein system, such as Bax, and the much simpler pore-forming antibiotic peptides.

Staphylococcus aureus bicomponent gamma-hemolysins, HlgA, HlgB, and HlgC, can form mixed pores containing all components.

Staphylococcal gamma-hemolysins are bicomponent toxins forming a protein family with leucocidins and alpha-toxin. Two active toxins (AB and CB) can be formed combining one of the class-S components, HlgA or HlgC, with the class-F component HlgB. These two gamma-hemolysins form pores with marked similarities to alpha-toxin in terms of conductance, nonlinearity of the current-voltage curve, and channel stability in the open state. AB and CB pores, however, are cation-selective, whereas alpha-toxin is anion-selective. gamma-Hemolysins' pores are hetero-oligomers formed by three or four copies of each component (indicated as 3A3B and 3C3B or 4A4B and 4C4B). Point mutants located on a beta-strand of the class-S component that forms part of the protomer-protomer contact region can prevent oligomer assembly. Interestingly, these mutants inhibit growth of pores formed not only by their natural components but also by nonstandard components. This lead to the hypothesis that mixed ABC pores could also be formed. By studying the conductance of pores, assembled in the presence of all three components (in different ratios), it was observed that the magnitudes expected for mixed pores were, indeed, present. We conclude that the gamma-hemolysin/leucocidin bicomponent toxin family may form a larger than expected number of active toxins by cross-combining various S and F components.

Peptides derived from apoptotic Bax and Bid reproduce the poration activity of the parent full-length proteins.

Bax and Bid are proapoptotic proteins of the Bcl-2 family that regulate the release of apoptogenic factors from mitochondria. Although they localize constitutively in the cytoplasm, their apoptotic function is exerted at the mitochondrial outer membrane, and is related to their ability to form transbilayer pores. Here we report the poration activity of fragments from these two proteins, containing the first alpha-helix of a colicinlike hydrophobic hairpin (alpha-helix 5 of Bax and alpha-helix 6 of Bid). Both peptides readily bind to synthetic lipid vesicles, where they adopt predominantly alpha-helical structures and induce the release of entrapped calcein. In planar lipid membranes they form ion conducting channels, which in the case of the Bax-derived peptide are characterized by a two-stage pattern, a large conductivity and lipid-charge-dependent ionic selectivity. These features, together with the influence of intrinsic lipid curvature on the poration activity and the existence of two helical stretches of different orientations for the membrane-bound peptide, suggest that it forms mixed lipidic/peptidic pores of toroidal structure. In contrast, the assayed Bid fragment shows a markedly different behavior, characterized by the formation of discrete, steplike channels in planar lipid bilayers, as expected for a peptidic pore lined by a bundle of helices.

Crystal structure of leucotoxin S component: new insight into the Staphylococcal beta-barrel pore-forming toxins.

Staphylococcal leucocidins and gamma-hemolysins (leucotoxins) are bi-component toxins that form lytic transmembrane pores. Their cytotoxic activities require the synergistic association of a class S component and a class F component, produced as water-soluble monomers that form hetero-oligomeric membrane-associated complexes. Strains that produce the Panton-Valentine leucocidin are clinically associated with cutaneous lesions and community-acquired pneumonia. In a previous study, we determined the crystal structure of the F monomer from the Panton-Valentine leucocidin. To derive information on the second component of the leucotoxins, the x-ray structure of the S protein from the Panton-Valentine leucocidin was solved to 2.0 angstrom resolution using a tetragonal crystal form that contains eight molecules in the asymmetric unit. The structure demonstrates the different conformation of the domain involved in membrane contacts and illustrates sequence and tertiary structure variabilities of the pore-forming leucotoxins. Mutagenesis studies at a key surface residue (Thr-28) further support the important role played by these microheterogeneities for the assembly of the bipartite leucotoxins.

Structure, conformation and biological activity of a novel lipodepsipeptide from Pseudomonas corrugata: cormycin A.

Cationic lipodepsipeptides from Pseudomonas spp. have been characterized for their structural and antimicrobial properties. In the present study, the structure of a novel lipodepsipeptide, cormycin A, produced in culture by the tomato pathogen Pseudomonas corrugata was elucidated by combined protein chemistry, mass spectrometry and two-dimensional NMR procedures. Its peptide moiety corresponds to L-Ser-D-Orn-L-Asn-D-Hse-L-His-L-aThr-Z-Dhb-L-Asp(3-OH)-L-Thr(4-Cl) [where Orn represents ornithine, Hse is homoserine, aThr is allo-threonine, Z-Dhb is 2,3-dehydro-2-aminobutanoic acid, Asp(3-OH) is 3-hydroxyaspartic acid and Thr(4-Cl) is 4-chlorothreonine], with the terminal carboxy group closing a macrocyclic ring with the hydroxy group of the N-terminal serine residue. This is, in turn, N-acylated by 3,4-dihydroxy-esadecanoate. In aqueous solution, cormycin A showed a rather compact structure, being derived from an inward orientation of some amino acid side chains and from the 'hairpin-bent' conformation of the lipid, due to inter-residue interactions involving its terminal part. Cormycin was significantly more active than the other lipodepsipeptides from Pseudomonas spp., as demonstrated by phytotoxicity and antibiosis assays, as well as by red-blood-cell lysis. Differences in biological activity were putatively ascribed to its weak positive net charge at neutral pH. Planar lipid membrane experiments showed step-like current transitions, suggesting that cormycin is able to form pores. This ability was strongly influenced by the phospholipid composition of the membrane and, in particular, by the presence of sterols. All of these findings suggest that cormycin derivatives could find promising applications, either as antifungal compounds for topical use or as post-harvest biocontrol agents.

Protein engineering modulates the transport properties and ion selectivity of the pores formed by staphylococcal gamma-haemolysins in lipid membranes.

Staphylococcal gamma-haemolysins are bicomponent toxins in a family including other leucocidins and alpha-toxin. Two active toxins are formed combining HlgA or HlgC with HlgB. Both open pores in lipid membranes with conductance, current voltage characteristics and stability similar to alpha-toxin, but different selectivity (cation instead of anion). Structural analogies between gamma-haemolysins and alpha-toxin indicate the presence, at the pore entry, of a conserved region containing four positive charges in alpha-toxin, but either positive or negative in gamma-haemolysins. Four mutants were produced (HlgA D44K, HlgB D47K, HlgB D49K and HlgB D47K/D49K) converting those negative charges to positive in HlgA and HlgB. When all charges were positive, the pores had the same selectivity and conductance as alpha-toxin, suggesting that the cluster may form an entrance electrostatic filter. As mutated HlgC-HlgB pores were less affected, additional charges in the lumen of the pore were changed (HlgB E107Q, HlgB D121N, HlgB T136D and HlgA K108T). Removing a negative charge from the lumen made the selectivity of both HlgA-HlgB D121N and HlgC-HlgB D121N more anionic. Residue D121 of HlgB is compensated by a positive residue (HlgA K108) in the HlgA-HlgB pore, but isolated in the more cation-selective HlgC-HlgB pore. Interestingly, the pore formed by HlgA K108T-HlgB, in which the positive charge of HlgA was removed, was as cation selective as HlgC-HlgB. Meanwhile, the pore formed by HlgA K108T-HlgB D121N, in which the two charge changes compensated, retrieved the properties of wild-type HlgA-HlgB. We conclude that the conductance and selectivity of the gamma-haemolysin pores depend substantially on the presence and location of charged residues in the channel.