Synergistic transmembrane insertion of the heterodimeric PGLa/magainin 2 complex studied by solid-state NMR

The skin secretions of amphibians are a rich source of antimicrobial peptides. The two antimicrobial peptides PGLa and magainin 2, isolated from the African frog Xenopus laevis, have been shown to act synergistically by permeabilizing the membranes of microorganisms. In this report, the literature on PGLa is extensively reviewed, with special focus on its synergistically enhanced activity in the presence of magainin 2. Our recent solid state ²H NMR studies of the orientation of PGLa in lipid membranes alone and in the presence of magainin 2 are described in detail, and some new data from 3,3,3-²H₃-L-alanine labeled PGLa are included in the analysis.     

Raman spectroscopy of synthetic antimicrobial frog peptides magainin 2a and PGLa

Magainin and PGLa are 23- and 21-residue peptides isolated from the skin of the African clawed frog Xenopus laevis. They protect the frog from infection and exhibit a broad-spectrum antimicrobial activity in vitro. The mechanism of this activity involves the interaction of magainin with microbial membranes. We have measured the secondary structure and membrane-perturbing ability of these peptides to obtain information about this mechanism. Our results show that mgn2a forms a helix with an average length of less than 20 A upon binding to liposomes. At high concentrations (50 mg/mL) mgn2a spontaneously solubilizes phosphatidylcholine liposomes at temperatures above the gel-liquid-crystalline phase transition. Mgn2a appears to bind to the surface of liposomes made of negatively charged lipids without spontaneously penetrating the bilayer. Finally, mgn2a and PGLa interact together with liposomes in a synergistic way that enhances the helix content of one or both of the peptides and allows the peptides to more easily penetrate the bilayer. PGLa mixed with a small nonperturbing amount of magainin 2 amide is 25-43 times as potent as PGLa alone at inducing the release of carboxyfluorescein from liposomes. The results suggest that the mechanism of antimicrobial activity does not involve a channel formed by transmembrane helical peptides.     

Heterodimer formation between the antimicrobial peptides magainin 2 and PGLa in lipid bilayers: a cross-linking study

The antimicrobial peptides magainin 2 and PGLa, isolated from the skin of the African clawed frog Xenopus laevis, show marked synergism [Westerhoff, H. V., Zasloff, M., Rosner, J. L., Hendler, R. W., de Waal, A., Vaz Gomes, A., Jongsma, A. P. M., Riethorst, A., and Juretic, D. (1995) Eur. J. Biochem. 228, 257-264]. We suggested previously that these peptides form a potent heterodimer composed of either parallel or antiparallel helices in membranes [Matsuzaki, K., Mitani, Y., Akada, K., Murase, O., Yoneyama, S., Zasloff, M., and Miyajima, K. (1998) Biochemistry 37, 15144-15153]. To detect the putative heterodimer by chemical cross-linking, analogues of magainin 2 and PGLa with a Cys residue at either terminus were synthesized. These cross-linking experiments suggested that both peptides form a parallel heterodimer in membranes composed of phosphatidylglycerol/phosphatidylcholine but not in either buffer or a helix-promoting 2,2,2-trifluoroethanol/buffer mixture. The isolated parallel heterodimers exhibited an order of magnitude higher membrane permeabilization activity compared with the monomeric species, indicating that the observed synergism is due to heterodimer formation.     

Membrane thickening by the antimicrobial peptide PGLa

Using x-ray diffraction, solid-state ²H-NMR, differential scanning calorimetry, and dilatometry, we have observed a perturbation of saturated acyl chain phosphatidylglycerol bilayers by the antimicrobial peptide peptidyl-glycylleucine-carboxyamide (PGLa) that is dependent on the length of the hydrocarbon chain. In the gel phase, PGLa induces a quasi-interdigitated phase, previously reported also for other peptides, which is most pronounced for C18 phosphatidylglycerol. In the fluid phase, we found an increase of the membrane thickness and NMR order parameter for C14 and C16 phosphatidylglycerol bilayers, though not for C18. The data is best understood in terms of a close hydrophobic match between the C18 bilayer core and the peptide length when PGLa is inserted with its helical axis normal to the bilayer surface. The C16 acyl chains appear to stretch to accommodate PGLa, whereas tilting within the bilayer seems to be energetically favorable for the peptide when inserted into bilayers of C14 phosphatidylglycerol. In contrast to the commonly accepted membrane thinning effect of antimicrobial peptides, the data demonstrate that pore formation does not necessarily relate to changes in the overall bilayer structure.     

Lipid-controlled peptide topology and interactions in bilayers: structural insights into the synergistic enhancement of the antimicrobial activities of PGLa and magainin 2

To gain further insight into the antimicrobial activities of cationic linear peptides, we investigated the topology of each of two peptides, PGLa and magainin 2, in oriented phospholipid bilayers in the presence and absence of the other peptide and as a function of the membrane lipid composition. Whereas proton-decoupled ¹⁵N solid-state NMR spectroscopy indicates that magainin 2 exhibits stable in-plane alignments under all conditions investigated, PGLa adopts a number of different membrane topologies with considerable variations in tilt angle. Hydrophobic thickness is an important parameter that modulates the alignment of PGLa. In equimolar mixtures of PGLa and magainin 2, the former adopts transmembrane orientations in dimyristoyl-, but not 1-palmitoyl-2-oleoyl-, phospholipid bilayers, whereas magainin 2 remains associated with the surface in all cases. These results have important consequences for the mechanistic models explaining synergistic activities of the peptide mixtures and will be discussed. The ensemble of data suggests that the thinning of the dimyristoyl membranes caused by magainin 2 tips the topological equilibrium of PGLa toward a membrane-inserted configuration. Therefore, lipid-mediated interactions play a fundamental role in determining the topology of membrane peptides and proteins and thereby, possibly, in regulating their activities as well.     

A two-dimensional NMR study of the antimicrobial peptide magainin 2

Using two-dimensional NMR spectroscopy, a complete 1H resonance assignment has been obtained for the peptide magainin 2 recently isolated from Xenopus laevis. It is demonstrated that this peptide adopts an alpha-helical structure with amphiphilic character when dissolved in a mixture of trifluoroethanol (TFE) and H2O. The transition to the alpha-helical conformation occurs at very low concentrations of TFE.     

Optimization of the antimicrobial activity of magainin peptides by modification of charge

Investigation of magainin II amide analogs with cationic charges ranging between +3 and +7 showed that enhancement of the peptide charge up to a threshold value of +5 and conservation of appropriate hydrophobic properties optimized the antimicrobial activity and selectivity. High selectivity was the result of both enhanced antimicrobial and reduced hemolytic activity. Charge increase beyond +5 with retention of other structural motifs led to a dramatic increase of hemolytic activity and loss of antimicrobial selectivity. Selectivity could be restored by reduction of the hydrophobicity of the hydrophobic helix surface (H(hd)), a structural parameter not previously considered to modulate activity. Dye release experiments with lipid vesicles revealed that the potential of peptide charge to modulate membrane activity is limited: on highly negatively charged 1-palmitoyl-2-oleoylphosphatidyl-DL-glycerol bilayers, reinforcement of electrostatic interactions had an activity-reducing effect. On neutral 1-palmitoyl-2-oleoylphosphatidylcholine bilayers, the high activity was determined by H(hd). H(hd) values above a certain threshold led to effective permeabilization of all lipid systems and even compensated for the activity-reducing effect of charge increase on highly negatively charged membranes.     

Crystallization of antimicrobial pores in membranes: magainin and protegrin

Membrane pores spontaneously formed by antimicrobial peptides in membranes were crystallized for the first time by manipulating the sample hydration and temperature. Neutron diffraction shows that magainins and protegrins form stable pores in fully hydrated fluid membranes. At lower hydration levels or low temperature, the membrane multilayers crystallize. In one crystalline phase, the pores in each bilayer arrange in a regular hexagonal array and the bilayers are stacked into a hexagonal ABC lattice, corresponding to the cubic close-packed structure of spheres. In another crystalline phase, the bilayers are modulated into the rippled multilamellae, corresponding to a 2D monoclinic lattice. The phase diagrams are described. Crystallization of the membrane pores provides possibilities for diffraction studies that might provide useful information on the pore structures.     

Concentration-dependent realignment of the antimicrobial peptide PGLa in lipid membranes observed by solid-state 19F-NMR

The membrane-disruptive antimicrobial peptide PGLa is found to change its orientation in a dimyristoyl-phosphatidylcholine bilayer when its concentration is increased to biologically active levels. The alignment of the alpha-helix was determined by highly sensitive solid-state NMR measurements of (19)F dipolar couplings on CF(3)-labeled side chains, and supported by a nonperturbing (15)N label. At a low peptide/lipid ratio of 1:200 the amphiphilic peptide resides on the membrane surface in the so-called S-state, as expected. However, at high peptide concentration (>/=1:50 molar ratio) the helix axis changes its tilt angle from approximately 90 degrees to approximately 120 degrees , with the C-terminus pointing toward the bilayer interior. This tilted "T-state" represents a novel feature of antimicrobial peptides, which is distinct from a membrane-inserted I-state. At intermediate concentration, PGLa is in exchange between the S- and T-state in the timescale of the NMR experiment. In both states the peptide molecules undergo fast rotation around the membrane normal in liquid crystalline bilayers; hence, large peptide aggregates do not form. Very likely the obliquely tilted T-state represents an antiparallel dimer of PGLa that is formed in the membrane at increasing concentration.     

Translocation of analogues of the antimicrobial peptides magainin and buforin across human cell membranes

Cationic antimicrobial peptides play important roles in innate immunity. Compared with extensive studies on peptide-bacteria interactions, little is known about peptide-human cell interactions. Using human cervical carcinoma HeLa and fibroblastic TM12 cells, we investigated the cellular uptake of fluorescent analogues of the two representative antimicrobial peptides magainin 2 and buforin 2 in comparison with the representative Arg-rich cell-penetrating Tat-(47-57) peptide (YGRKKRRQRRR). The dose, time, temperature, and energy dependence of translocation suggested that the three peptides cross cell membranes through different mechanisms. The magainin peptide was internalized within a time scale of tens of minutes. The cooperative concentration dependence of uptake suggested that the peptide forms a pore as an intermediate similar to the observations in model membranes. Furthermore, the translocation was coupled with cytotoxicity, which was larger for tumor HeLa cells. In contrast, the buforin peptide translocated within 10 min by a temperature-independent, less concentration-dependent passive mechanism without showing any significant cytotoxicity at the highest concentration investigated (100 microm). The uptake of the Tat peptide was proportional to the peptide concentration, and the concentration dependence was lost upon ATP depletion. The peptide exhibited a moderate cytotoxicity at higher concentrations. The time course did not show saturation even after 120 min. The buforin peptide, covalently attached to the 28-kDa green fluorescent protein, also entered cells, suggesting a potency of the peptide as a vector for macromolecular delivery into cells. However, the mechanism appeared to be different from that of the parent peptide.     

Grazing incidence X-ray diffraction of highly aligned phospholipid membranes containing the antimicrobial peptide magainin 2

We present the first study of grazing incidence X-ray diffraction on a model system of phospholipid membranes and antimicrobial peptides. For this purpose, highly oriented multilamellar samples have been prepared on solid substrates. By this technique, the short-range order of the lipid chains in the fluid L_α phase can be investigated quantitatively, including not only the mean distance between acyl chains, but also the associated correlation length. The short-range order in lecithin is found to be severely affected by the amphiphilic peptide magainin 2. Received: 7 June 1999 / Revised version: 6 September 1999 / Accepted: 17 September 1999     

Synergistic antibacterial action of heat in combination with nisin and magainin II amide

Lactobacillus plantarum has been exposed to mild heat at temperatures between 48 and 56 °C in combination with low concentrations of the lantobiotic nisin in different sequential set-ups. Exposure to heat and nisin caused synergistic reductions of Lact. plantarum viability. Efficient antimicrobial action was dependent on the growth state of the culture as well as on levels and sequences of treatment applications. Listeria monocytogenes and Escherichia coli were treated at 55 °C in the presence of magainin II amide. Synergistic reductions in viable counts could be observed for L. monocytogenes and, after prolonged exposure, also for E. coli . The bacterial membrane could be identified by fluorometry and flow cytometry as an important target of applied treatment combinations.     

Interactions between Salmonella typhimurium lipopolysaccharide and the antimicrobial peptide, magainin 2 amide

Effects of magainin 2 amide on the phase behavior of Salmonella typhimurium lipopolysaccharide were characterized by FT-IR spectroscopy. This antimicrobial cationic peptide disorders the lipopolysaccharide at molecular ratios of lipopolysaccharide to magainin greater than 4, and can induce a temperature-dependent structural reorientation. The nature of the five phosphate groups of lipopolysaccharide was determined by 31P NMR spectroscopy. At pH 7.4, the net charge on the phosphates is -7. Lipopolysaccharide undoubtedly plays an important role in modulating the interactions of magainin with the gram-negative cell envelope and may act as a molecular sponge to protect the plasma membrane.     

Comparison of the membrane association of two antimicrobial peptides, magainin 2 and indolicidin

Interactions of two antimicrobial peptides, magainin 2 and indolicidin, with three different model biomembranes, namely, monolayers, large unilamellar vesicles (LUVs), and giant liposomes, were studied. Insertion of both peptides into lipid monolayers was progressively enhanced when the content of an acidic phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) in a film of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) was increased. Indolicidin and magainin 2 penetrated also into lipid monolayers containing cholesterol (mole fraction, X = 0.1). Membrane association of magainin 2 attenuated lipid lateral diffusion in POPG-containing LUVs as revealed by the decrease in the excimer/monomer fluorescence ratio I(e)/I(m) for the pyrene fatty-acid-containing phospholipid derivative 1-palmitoyl-2-[10-(pyren-1-yl) decanoyl]-sn-glycero-3-phospho-rac-glycerol (PPDPG). Likewise, an increase in steady-state fluorescence anisotropy of the membrane-incorporated diphenylhexatriene (DPH) was observed, revealing magainin 2 to increase acyl chain order and induce segregation of acidic phospholipids. Similar effects were observed for indolicidin. The topological effects of magainin 2 and indolicidin on phospholipid membranes were investigated using optical microscopy of giant vesicles. Magainin 2 had essentially no influence on either SOPC or SOPC:cholesterol (X = 0.1) giant liposomes. However, effective vesiculation was observed when acidic phospholipid (X(PG) = 0.1) was included in the giant vesicles. Indolicidin caused only a minor shrinkage of giant SOPC vesicles whereas the formation of endocytotic vesicles was observed when the giant liposome contained POPG (X(PG) = 0.1). Interestingly, for indolicidin, vesiculation was also observed for giant vesicles composed of SOPC/cholesterol (X(chol) = 0.1). Possible mechanisms of membrane transformation induced by these two peptides are discussed.     

Conditions affecting the re-alignment of the antimicrobial peptide PGLa in membranes as monitored by solid state H-NMR

The cationic antimicrobial peptide PGLa is electrostatically attracted to bacterial membranes, binds as an amphiphilic α-helix, and is thus able to permeabilize the lipid bilayer. Using solid state ²H-NMR of non-perturbing Ala-d₃ labels on the peptide, we have characterized the helix alignment under a range of different conditions. Even at a very high peptide-to-lipid ratio (1:20) and in the presence of negatively charged lipids, there was no indication of a toroidal wormhole structure. Instead, PGLa re-aligns from a surface-bound S-state to an obliquely tilted T-state, which is presumably dimeric. An intermediate structure half-way between the S- and T-state was observed in fully hydrated multilamellar DMPC vesicles at 1:50, suggesting a fast exchange between the two states on the time scale of >50 kHz. We demonstrate that this equilibrium is shifted from the S- towards the T-state either upon (i) increasing the peptide concentration, (ii) adding negatively charged DMPG, or (iii) decreasing the level of hydration. The threshold concentration for re-alignment in DMPC is found to be between 1:200 and 1:100 in oriented samples at 96% humidity. In fully hydrated multilamellar DMPC vesicles, it shifts to an effective peptide-to-lipid ratio of 1:50 as some peptides are able to escape into the bulk water phase.     

Unambiguous prediction of human integrin transmembrane heterodimer interactions using only homologous sequences

Part of the interaction between the alpha- and beta-subunits of integrins is known to take place at the transmembrane (TM) domain, where both heteromeric and homomeric aggregates have been reported in vivo and in vitro. In a recent computational study, totally independent from biochemical or biophysical data, we explored the plausibility of various TM homo-oligomers using evolutionary conservation data as a filter for non-native interactions. We showed that several homodimeric and homotrimeric interactions for alpha- and beta-chains are evolutionarily conserved. We report herein the results of the application of the same exhaustive approach to the integrin heterodimer. We have studied all known human TM integrin alphabeta pairs, and we show unambiguously that two models of interaction are evolutionarily conserved. These two models are consistent with those proposed previously based on mutagenesis and crosslinking. Comparison with previous experimental data strongly supports that a glycophorin A-like model is an intermediate form of interaction between the resting state and the active form, where chain separation occurs. Surprisingly, these two models are also conserved when considering most of the possible alphabeta pair combinations, suggesting that specific pairing of integrins is not determined by the TM domain, which has remained unchanged in spite of the variety of known integrin functions. This fact highlights a common ancestral mechanism for signal transduction that has remained through evolution. In a broader context, our results show that it is possible to obtain correct and detailed interactions of alpha-helical heterodimers with total independence of experimental data.     

Position-dependent hydrophobicity of the antimicrobial magainin peptide affects the mode of peptide-lipid interactions and selective toxicity

Cationic antimicrobial peptides are promising candidates as novel antibiotics of clinical usefulness. Magainin 2, a representative antimicrobial peptide isolated from the skin of the African clawed frog Xenopus leavis, electrostatically recognizes anionic lipids that are abundant in bacterial membranes, forming a peptide-lipid supramolecular complex pore, whereas the peptide does not effectively bind to zwitterionic phospholipids constituting the outer leaflets of mammalian cell membranes because of the low hydrophobicity of the peptide [Matsuzaki, K. (1999) Biochim. Biophys. Acta 1462, 1-10]. In this study, two magainin analogues with enhanced hydrophobicity, MG-H1 (GIKKFLHIIWKFIKAFVGEIMNS) and MG-H2 (IIKKFLHSIWKFGKAFVGEIMNI), with identical amino acid compositions were designed and interactions with lipid bilayers and biological activities were examined in comparison with those of MG (GIGKWLHSAKKFGKAFVGEIMNS = F5W-magainin 2). The apparent hydrophobicities and hydrophobic moments of MG-H1 and MG-H2, conventionally calculated assuming that all residues are involved in helix formation, were almost the same. MG-H2 behaved like MG except for greatly enhanced activity against zwitterionic membranes and erythrocytes. In contrast, despite a very similar calculated hydrophobicity, the observed hydrophobicity of MG-H1 was larger than that of MG-H2 because of a tendency toward helix fraying near the termini. Therefore, the physicochemical parameters of only the helical portion should be considered in characterizing peptide-lipid interactions, although this point was overlooked in most studies. Moreover, MG-H1 induced aggregation and/or fusion of negatively charged membranes. Furthermore, the peptide hydrophobicity was found to affect pore formation rate, pore size, and pore stability. These observations demonstrate that the hydrophobicity of the peptide also controls the mode of action and is dependent on the position of the hydrophobic amino acids in the peptide sequence.     

Conditions affecting the re-alignment of the antimicrobial peptide PGLa in membranes as monitored by solid state 2H-NMR

The cationic antimicrobial peptide PGLa is electrostatically attracted to bacterial membranes, binds as an amphiphilic alpha-helix, and is thus able to permeabilize the lipid bilayer. Using solid state (2)H-NMR of non-perturbing Ala-d(3) labels on the peptide, we have characterized the helix alignment under a range of different conditions. Even at a very high peptide-to-lipid ratio (1:20) and in the presence of negatively charged lipids, there was no indication of a toroidal wormhole structure. Instead, PGLa re-aligns from a surface-bound S-state to an obliquely tilted T-state, which is presumably dimeric. An intermediate structure half-way between the S- and T-state was observed in fully hydrated multilamellar DMPC vesicles at 1:50, suggesting a fast exchange between the two states on the time scale of >50 kHz. We demonstrate that this equilibrium is shifted from the S- towards the T-state either upon (i) increasing the peptide concentration, (ii) adding negatively charged DMPG, or (iii) decreasing the level of hydration. The threshold concentration for re-alignment in DMPC is found to be between 1:200 and 1:100 in oriented samples at 96% humidity. In fully hydrated multilamellar DMPC vesicles, it shifts to an effective peptide-to-lipid ratio of 1:50 as some peptides are able to escape into the bulk water phase.