Biophysical studies of the interactions between the phage ϕKZ gp144 lytic transglycosylase and model membranes

The use of naturally occurring lytic bacteriophage proteins as specific antibacterial agents is a promising way to treat bacterial infections caused by antibiotic-resistant pathogens. The opportunity to develop bacterial resistance to these agents is minimized by their broad mechanism of action on bacterial membranes and peptidoglycan integrity. In the present study, we have investigated lipid interactions of the gp144 lytic transglycosylase from the Pseudomonas aeruginosa phage ϕKZ. Interactions with zwitterionic lipids characteristic of eukaryotic cells and with anionic lipids characteristic of bacterial cells were studied using fluorescence, solid-state nuclear magnetic resonance, Fourier transform infrared, circular dichroism, Langmuir monolayers, and Brewster angle microscopy (BAM). Gp144 interacted preferentially with anionic lipids, and the presence of gp144 in anionic model systems induced membrane disruption and lysis. Lipid domain formation in anionic membranes was observed by BAM. Gp144 did not induce disruption of zwitterionic membranes but caused an increase in rigidity of the lipid polar head group. However, gp144 interacted with zwitterionic and anionic lipids in a model membrane system containing both lipids. Finally, the gp144 secondary structure was not significantly modified upon lipid binding.     

A spectroscopic study of the membrane interaction of the antimicrobial peptide Pleurocidin

The cationic amphipathic alpha-helical antibiotic peptide, pleurocidin, from the winter flounder Pleuronectes americanus associates strongly with anionic membranes where it is able to translocate across the membrane, cause dye leakage from vesicles and induce pore like channel conductance. To investigate the mechanism of pleurocidin antibiotic activity in more detail we have applied a variety of spectroscopic techniques to study the interaction of pleurocidin with model membranes. At neutral pH the peptide inserts into membranes containing anionic lipids and, as shown by proton-decoupled 15N solid-state NMR spectroscopy of macroscopically oriented samples, is aligned parallel to the membrane surface. 2H solid-state NMR spectroscopy of chain deuterated phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) lipids in mixed membranes shows that pleurocidin interacts with both the zwitterionic PE and anionic PG but disrupts the lipid acyl chain order of the anionic PG lipids more effectively. At acidic pH the three histidine residues of pleurocidin become protonated and positively charged which does not alter the membrane disrupting effect nor the location of the peptide in the membrane. The results are interpreted in terms of a structural model for pleurocidin inserted into anionic lipid membranes and the implications of our data are discussed in terms of a general mechanism for the antibiotic activity.     

Interaction of diphtheria toxin with model membranes

Low pH is believed to trigger membrane penetration by diphtheria toxin in vivo. The effect of pH upon the binding of the toxin to unilamellar model membrane vesicles was determined by using a fluorescence quenching assay. A series of studies were undertaken to determine the effect of lipid composition upon the binding of lipids to the toxin. The binding of toxin to various small unilamellar vesicles of zwitterionic or anionic lipids was similar in extent and was accompanied by deep penetration of the toxin into the fatty acyl chains, in agreement with previous studies. However, the transition pH, which is the pH at and below which toxin binding becomes significant, depended upon the fraction of anionic lipids, being highest with model membranes composed totally of anionic lipids (pH 5.8) and lowest with membranes composed of zwitterionic lipids (pH 5.2). Except for vesicle charge, the transition pH was independent of the nature of the lipid polar groups used. High ionic strength, which had no effect on the transition pH with zwitterionic vesicles, was found to shift the transition pH with totally anionic vesicles to pH 5.2. This suggests that both direct protein-lipid electrostatic interactions and the ionic double layer, which gives rise to a low local pH around anionic vesicles, contribute to the shift in the transition pH. The effect of lipid composition upon the kinetics and strength of binding was also examined. At low pH, binding was rapid and tight. Binding to vesicles containing 20 wt % anionic phosphatidylglycerol was faster and tighter than binding to vesicles of zwitterionic phosphatidylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biophysical Investigation of the Membrane-Disrupting Mechanism of the Antimicrobial and Amyloid-Like Peptide Dermaseptin S9

Dermaseptin S9 (Drs S9) is an atypical cationic antimicrobial peptide with a long hydrophobic core and with a propensity to form amyloid-like fibrils. Here we investigated its membrane interaction using a variety of biophysical techniques. Rather surprisingly, we found that Drs S9 induces efficient permeabilisation in zwitterionic phosphatidylcholine (PC) vesicles, but not in anionic phosphatidylglycerol (PG) vesicles. We also found that the peptide inserts more efficiently in PC than in PG monolayers. Therefore, electrostatic interactions between the cationic Drs S9 and anionic membranes cannot explain the selectivity of the peptide towards bacterial membranes. CD spectroscopy, electron microscopy and ThT fluorescence experiments showed that the peptide adopts slightly more β-sheet and has a higher tendency to form amyloid-like fibrils in the presence of PC membranes as compared to PG membranes. Thus, induction of leakage may be related to peptide aggregation. The use of a pre-incorporation protocol to reduce peptide/peptide interactions characteristic of aggregates in solution resulted in more α-helix formation and a more pronounced effect on the cooperativity of the gel-fluid lipid phase transition in all lipid systems tested. Calorimetric data together with ²H- and ³¹P-NMR experiments indicated that the peptide has a significant impact on the dynamic organization of lipid bilayers, albeit slightly less for zwitterionic than for anionic membranes. Taken together, our data suggest that in particular in membranes of zwitterionic lipids the peptide binds in an aggregated state resulting in membrane leakage. We propose that also the antimicrobial activity of Drs S9 may be a result of binding of the peptide in an aggregated state, but that specific binding and aggregation to bacterial membranes is regulated not by anionic lipids but by as yet unknown factors.     

Minimal Effect of Lipid Charge on Membrane Miscibility Phase Behavior in Three Ternary Systems

Giant unilamellar vesicles composed of a ternary mixture of phospholipids and cholesterol exhibit coexisting liquid phases over a range of temperatures and compositions. A significant fraction of lipids in biological membranes are charged. Here, we present phase diagrams of vesicles composed of phosphatidylcholine (PC) lipids, which are zwitterionic; phosphatidylglycerol (PG) lipids, which are anionic; and cholesterol (Chol). Specifically, we use DiPhyPG-DPPC-Chol and DiPhyPC-DPPG-Chol. We show that miscibility in membranes containing charged PG lipids occurs over similarly high temperatures and broad lipid compositions as in corresponding membranes containing only uncharged lipids, and that the presence of salt has a minimal effect. We verified our results in two ways. First, we used mass spectrometry to ensure that charged PC/PG/Chol vesicles formed by gentle hydration have the same composition as the lipid stocks from which they are made. Second, we repeated the experiments by substituting phosphatidylserine for PG as the charged lipid and observed similar phenomena. Our results consistently support the view that monovalent charged lipids have only a minimal effect on lipid miscibility phase behavior in our system.     

Analytic Approaches to Stochastic Gene Expression in Multicellular Systems

Giant unilamellar vesicles composed of a ternary mixture of phospholipids and cholesterol exhibit coexisting liquid phases over a range of temperatures and compositions. A significant fraction of lipids in biological membranes are charged. Here, we present phase diagrams of vesicles composed of phosphatidylcholine (PC) lipids, which are zwitterionic; phosphatidylglycerol (PG) lipids, which are anionic; and cholesterol (Chol). Specifically, we use DiPhyPG-DPPC-Chol and DiPhyPC-DPPG-Chol. We show that miscibility in membranes containing charged PG lipids occurs over similarly high temperatures and broad lipid compositions as in corresponding membranes containing only uncharged lipids, and that the presence of salt has a minimal effect. We verified our results in two ways. First, we used mass spectrometry to ensure that charged PC/PG/Chol vesicles formed by gentle hydration have the same composition as the lipid stocks from which they are made. Second, we repeated the experiments by substituting phosphatidylserine for PG as the charged lipid and observed similar phenomena. Our results consistently support the view that monovalent charged lipids have only a minimal effect on lipid miscibility phase behavior in our system.     

Investigating the interfacial binding of bacterial phosphatidylinositol-specific phospholipase C

The interactions of PI-PLC with nonsubstrate zwitterionic [phosphatidylcholine (PC)] and anionic [phosphatidylmethanol (PMe), phosphatidylserine, phosphatidylglycerol, and phosphatidic acid] interfaces that affect the catalytic activity of PI-PLC have been examined. PI-PLC binding is strongly coupled to vesicle curvature and is tighter at acidic pH for all of the phospholipids examined. PI-PLC binds to small unilamellar vesicles (SUVs) of anionic lipids with much higher affinity (K(d) is 0.01-0.07 microM for a site consisting of n = 100 +/- 25 lipids when analyzed with a Langmuir adsorption isotherm) than to zwitterionic PC SUVs (K(d) is 5-20 microM and n = 8 +/- 3). The binding to PC surfaces is dominated by hydrophobic interactions, while binding to anionic surfaces is dominated by electrostatic interactions. The contributions of specific cationic side chains and hydrophobic groups at the rim of the alpha beta-barrel to zwitterionic and anionic vesicle binding have been assessed with mutagenesis. The results are used to explain how PC activates the enzyme for both phosphotransferase and cyclic phosphodiesterase activities.     

Interaction of the K+ channel KcsA with membrane phospholipids as studied by ESI mass spectrometry

In this study we have used electrospray ionization mass spectrometry (ESI-MS) to investigate interactions between the bacterial K(+) channel KcsA and membrane phospholipids. KcsA was reconstituted into lipid vesicles of variable lipid composition. These vesicles were directly analyzed by ESI-MS or mixed with trifluoroethanol (TFE) before analysis. In the resulting mass spectra, non-covalent complexes of KcsA and phospholipids were observed with an interesting lipid specificity. The anionic phosphatidylglycerol (PG), and, to a lesser extent, the zwitterionic phosphatidylethanolamine (PE), which both are abundant bacterial lipids, were found to preferentially associate with KcsA as compared to the zwitterionic phosphatidylcholine (PC). These preferred interactions may reflect the differences in affinity of these phospholipids for KcsA in the membrane.     

Magainin 2 Revisited: A Test of the Quantitative Model for the All-or-None Permeabilization of Phospholipid Vesicles

The all-or-none kinetic model that we recently proposed for the antimicrobial peptide cecropin A is tested here for magainin 2. In mixtures of phosphatidylcholine (PC)/phosphatidylglycerol (PG) 50:50 and 70:30, release of contents from lipid vesicles occurs in an all-or-none fashion and the differences between PC/PG 50:50 and 70:30 can be ascribed mainly to differences in binding, which was determined independently and is ∼20 times greater to PC/PG 50:50 than to 70:30. Only one variable parameter, β, corresponding to the ratio of the rates of pore opening to pore closing, is used to fit dye release kinetics from these two mixtures, for several peptide/lipid ratios ranging from 1:25 to 1:200. However, unlike for cecropin A where it stays almost constant, β increases five times as the PG content of the vesicles increases from 30 to 50%. Thus, magainin 2 is more sensitive to anionic lipid content than cecropin A. But overall, magainin follows the same all-or-none kinetic model as cecropin A in these lipid mixtures, with slightly different parameter values. When the PG content is reduced to 20 mol %, dye release becomes very low; the mechanism appears to change, and is consistent with a graded kinetic model. We suggest that the peptide may be inducing formation of PG domains. In either mechanism, no peptide oligomerization occurs and magainin catalyzes dye release in proportion to its concentration on the membrane in a peptide state that we call a pore. We envision this structure as a chaotic or stochastic type of pore, involving both lipids and peptides, not a well-defined, peptide-lined channel.     

Channel formation properties of synthetic pardaxin and analogues.

Six analogues of teh 33-residue shark repellent neurotoxin pardaxin were synthesized by the solid phase method: [Ala13]pardaxin, [Gly14,Gly15]pardaxin, des[1----9]pardaxin, [N1-succinamido]pardaxin, C33-dihydroxyethylamido]pardaxin, and C33-[diaminoethylamido]pardaxin. The spectroscopic and functional characterizations of the analogues are described. The peptides were characterized spectroscopically by circular dichroism (CD) before and after binding to soybean vesicles. They were characterized functionally by measuring their potential to evoke the dissipation of diffusion potential and calcein release from sonicated unilamellar soybean liposomes, by determining their ability to create single channels in planar bilayers, and by measuring their cytolytic activity on human erythrocytes. The behavior of the analogues modified at the C terminus is similar to that of pardaxin. [N'-succinamido]Pardaxin, however, reveals an increase in alpha-helicity both alone and in the presence of liposomes. It has the same potency as pardaxin to dissipate diffusion potential, to evoke calcein release and to produce single channels in lipid bilayers, but at a slower rate than that of pardaxin. It has more than 70-fold less cytolytic activity than pardaxin. [Ala13] Pardaxin has twice the alpha-helical content than pardaxin, both alone and in the presence of vesicles, yet it has less effect on the diffusion potential and calcein release, and it does not have cytolytic activity on human erythrocytes. Both [Gly14,Gly15]pardaxin and des[1----9]pardaxin are much less potent than pardaxin in all effects. However des[1----9]pardaxin exhibits a slight change in alpha-helicity upon binding to vesicles, whereas [Gly14,Gly15]pardaxin does not. The results support a model in which pardaxin is composed of two putative alpha-helices separated by proline. The N-terminal alpha-helix is important for the insertion of the peptide to the lipid bilayer, and the C-terminal amphiphilic alpha-helix is the ion channel lining segment of pardaxin.     

Membrane fusion by an RGD-containing sequence from the core protein VP3 of hepatitis A virus and the RGA-analogue: implications for viral infection

The interaction of an RGD-containing epitope from the hepatitis A virus VP3 capsid protein and its RGA-analogue with lipid membranes was studied by biophysical methods. Two types of model membrane were used: vesicles and monolayers spread at the air/water interface, with a composition that closely resembles the lipid moiety of hepatocyte membranes: PC/SM/PE/PC (40:33:12:15; PC: 1-palmitoyl-2-oleoylglycero-sn-3-phosphocholine; SM: sphingomyelin from chicken egg yolk; PE, 1,2-dipalmitoyl-phosphatidylethanolamine; PS: L-alpha-phosphatidyl-L-serine from bovine brain). In addition, zwitterionic PC/SM/PE (47:39:14) and cationic PC/SM/PE/DOTAP (40:33:12:15; DOTAP: 1,2-dioleoyl-3-trimethylammonium-propane) membranes were also prepared in order to dissect the electrostatic and hydrophobic components in the interaction. Changes in tryptophan fluorescence, acrylamide quenching, and resonance energy transfer experiments in the presence of vesicles, as well as the kinetics of insertion in monolayers, indicate that both peptides bind to the three types of membrane at neutral and acidic pH; however, binding is irreversible only at low pH. Membrane-destabilizing and fusogenic activities are triggered by acidification at pH 4-6, characteristic of the endosome. Fluorescence experiments show that VP3-RGD and VP3-RGA induce mixing of lipids and leakage or mixing of aqueous contents in anionic and cationic vesicles at pH 4-6, indicating leaky fusion. Interaction with zwitterionic vesicles (PC/SM/PE) results in leakage without lipid mixing, indicating pore formation. Replacement of aspartic acid in the RGD motif by alanine maintains the membrane-destabilizing properties of the peptide at low pH, but not its antigenicity. Since the RGD tripeptide is related to receptor-mediated cell adhesion and antigenicity, results suggest that receptor binding is not a molecular requirement for fusion. The possible involvement of peptide-induced membrane destabilization in the mechanism of hepatitis A virus infection of hepatocytes by the endosomal route is discussed.     

Effects of Lipid Structure on the State of Aggregation of Potassium Channel KcsA

The state of aggregation of potassium channel KcsA was determined as a function of lipid:protein molar ratio in bilayer membranes of the zwitterionic lipid phosphatidylcholine (PC) and of the anionic lipid phosphatidylglycerol (PG). EPR (electron paramagnetic resonance) with spin-labeled phospholipids was used to determine the number of motionally restricted lipids per KcsA tetramer. Unexpectedly, this number decreased with a decreasing lipid:KcsA tetramer molar ratio in the range of 88:1 to 30:1, consistent with sharing of annular lipid shells and KcsA-KcsA contact at high mole fractions of protein. Fluorescence quenching experiments with brominated phospholipids showed a decrease in fluorescence quenching at low lipid:KcsA tetramer mole ratios, also consistent with KcsA-KcsA contact at high mole fractions of protein. The effects of low mole ratios of lipid seen in EPR and fluorescence quenching experiments were more marked in bilayers of PC than in bilayers of PG, suggesting stronger association of PG than PC with KcsA. This was confirmed by direct measurement of lipid association constants using spin-labeled phospholipids, showing higher association constants for all anionic lipids than for PC. The results show that the probability of contacts between KcsA tetramers will be very low at lipid:protein molar ratios that are typical of native biological membranes.     

CTP:phosphocholine cytidylyltransferase binds anionic phospholipid vesicles in a cross-bridging mode

CTP:phosphocholine cytidylyltransferase (CCT) catalyzes the rate-limiting step in phosphatidylcholine (PC) synthesis, and its activity is regulated by reversible association with membranes, mediated by an amphipathic helical domain M. Here we describe a new feature of the CCTalpha isoform, vesicle tethering. We show, using dynamic light scattering and transmission electron microscopy, that dimers of CCTalpha can cross-bridge separate vesicles to promote vesicle aggregation. The vesicles contained either class I activators (anionic phospholipids) or the less potent class II activators, which favor nonlamellar phase formation. CCT increased the apparent hydrodynamic radius and polydispersity of anionic phospholipid vesicles even at low CCT concentrations corresponding to only one or two dimers per vesicle. Electron micrographs of negatively stained phosphatidylglycerol (PG) vesicles confirmed CCT-mediated vesicle aggregation. CCT conjugated to colloidal gold accumulated on the vesicle surfaces and in areas of vesicle-vesicle contact. PG vesicle aggregation required both the membrane-binding domain and the intact CCT dimer, suggesting binding of CCT to apposed membranes via the two M domains situated on opposite sides of the dimerization domain. In contrast to the effects on anionic phospholipid vesicles, CCT did not induce aggregation of PC vesicles containing the class II lipids, oleic acid, diacylglycerol, or phosphatidylethanolamine. The different behavior of the two lipid classes reflected differences in measured binding affinity, with only strongly binding phospholipid vesicles being susceptible to CCT-induced aggregation. Our findings suggest a new model for CCTalpha domain organization and membrane interaction, and a potential involvement of the enzyme in cellular events that implicate close apposition of membranes.     

Activity and characterization of a pH-sensitive antimicrobial peptide

Antimicrobial peptides (AMPs) have been an area of great interest, due to the high selectivity of these molecules toward bacterial targets over host cells and the limited development of bacterial resistance to these molecules throughout evolution. Previous work showed that when Histidine was incorporated into the peptide C18G it lost antimicrobial activity. The role of pH on activity and biophysical properties of the peptide was investigated to explain this phenomenon. Minimal inhibitory concentration (MIC) results demonstrated that decreased media pH increased antimicrobial activity. Trichloroethanol (TCE) quenching and red-edge excitation spectroscopy (REES) showed a clear pH dependence on peptide aggregation in solution. Trp fluorescence was used to monitor binding to lipid vesicles and demonstrated the peptide binds to anionic bilayers at all pH values tested, however, binding to zwitterionic bilayers was enhanced at pH 7 and 8 (above the His pKa). Dual Quencher Analysis (DQA) confirmed the peptide inserted more deeply in PC:PG and PE:PG membranes, but could insert into PC bilayers at pH conditions above the His pKa. Bacterial membrane permeabilization assays which showed enhanced membrane permeabilization at pH 5 and 6 but vesicle leakage assays indicate enhanced permeabilization of PC and PC:PG bilayers at neutral pH. The results indicate the ionization of the His side chain affects the aggregation state of the peptide in solution and the conformation the peptide adopts when bound to bilayers, but there are likely more subtle influences of lipid composition and properties that impact the ability of the peptide to form pores in membranes.     

Cholesterol and anionic phospholipids increase the binding of amyloidogenic transthyretin to lipid membranes

Deposition of transthyretin (TTR) amyloid is a pathological hallmark of familial amyloidotic polyneuropathy (FAP). Recently we showed that TTR binds to membrane lipids via electrostatic interactions and that membrane binding is correlated with the cytotoxicity induced by amyloidogenic TTR. In the present study, we examined the role of lipid composition in membrane binding of TTR by a surface plasmon resonance (SPR) approach. TTR bound to lipid bilayers through both high- and low-affinity interactions. Increasing the mole fraction of cholesterol in the bilayer led to an increase in the amount of high-affinity binding of an amyloidogenic mutant (L55P) TTR. In addition, a greater amount of L55P TTR bound with high affinity to membranes made from anionic phospholipids, phosphatidylglycerol (PG) and phosphatidylserine (PS), than to membranes made from zwitterionic phospholipid phosphatidylcholine (PC). The anionic phospholipids (PS and PG) promoted the aggregation of L55P TTR by accelerating the nucleation phase of aggregation, whereas the zwitterionic phospholipid PC had little effect. These results suggest that cholesterol and anionic phospholipids may be important for TTR aggregation and TTR-induced cytotoxicity.     

Cholesterol and anionic phospholipids increase the binding of amyloidogenic transthyretin to lipid membranes

Deposition of transthyretin (TTR) amyloid is a pathological hallmark of familial amyloidotic polyneuropathy (FAP). Recently we showed that TTR binds to membrane lipids via electrostatic interactions and that membrane binding is correlated with the cytotoxicity induced by amyloidogenic TTR. In the present study, we examined the role of lipid composition in membrane binding of TTR by a surface plasmon resonance (SPR) approach. TTR bound to lipid bilayers through both high- and low-affinity interactions. Increasing the mole fraction of cholesterol in the bilayer led to an increase in the amount of high-affinity binding of an amyloidogenic mutant (L55P) TTR. In addition, a greater amount of L55P TTR bound with high affinity to membranes made from anionic phospholipids, phosphatidylglycerol (PG) and phosphatidylserine (PS), than to membranes made from zwitterionic phospholipid phosphatidylcholine (PC). The anionic phospholipids (PS and PG) promoted the aggregation of L55P TTR by accelerating the nucleation phase of aggregation, whereas the zwitterionic phospholipid PC had little effect. These results suggest that cholesterol and anionic phospholipids may be important for TTR aggregation and TTR-induced cytotoxicity.     

Interfacial catalysis by phospholipase A2: substrate specificity in vesicles.

The binding equilibrium of phospholipase A2 (PLA2) to the substrate interface influences many aspects of the overall kinetics of interfacial catalysis by this enzyme. For example, the interpretation of kinetic data on substrate specificity was difficult when there was a significant kinetic contribution from the interfacial binding step to the steady-state catalytic turnover. This problem was commonly encountered with vesicles of zwitterionic phospholipids, where the binding of PLA2 to the interface was relatively poor. The action of PLA2 on phosphatidylcholine (PC) vesicles containing a small amount of anionic phospholipid, such as phosphatidic acid (PA), was studied. It was shown that the hydrolysis of these mixed lipid vesicles occurs in the scooting mode in which the enzyme remains tightly bound to the interface and only the substrate molecules present on the outer monolayer of the target vesicle became hydrolyzed Thus the phenomenon of scooting mode hydrolysis was not restricted to the action of PLA2 on vesicles of pure anionic phospholipids, but it was also observed with vesicles of zwitterionic lipids as long as a critical amount of anionic compound was present. Under such conditions, the initial rate of hydrolysis of PC in the mixed PC/PA vesicles was enhanced more than 50-fold. Binding studies of PLA2 to vesicles and kinetic studies in the scooting mode demonstrated that the enhancement of PC hydrolysis in the PC/PA covesicles was due to the much higher affinity of the enzyme toward covesicles compared to vesicles of pure PC phospholipids. A novel and technically simple protocol for accurate determination of the substrate specificity of PLA2 at the interface was also developed by using a double-radiolabel approach. Here, the action of PLA2 in the scooting mode was studied on vesicles of the anionic phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphomethanol that contained small amounts of 3H- and 14C-labeled phospholipids. From an analysis of the 3H and 14C radioactivity in the released fatty acid products, the ratio of substrate specificity constants (kcat/KMS) was obtained for any pair of radiolabeled substrates. These studies showed that the PLA2s from pig pancreas and Naja naja naja venom did not discriminate between phosphatidylcholine and phosphatidylethanolamine phospholipids or between phospholipids with saturated versus unsaturated acyl chains and that the pig enzyme had a slight preference for anionic phospholipids (2-3-fold). The described protocol provided an accurate measure of the substrate specificity of PLA2 without complications arising from the differences in binding affinities of the enzyme to vesicles composed of pure phospholipids.     

Cytoplasmic residues of phospholamban interact with membrane surfaces in the presence of SERCA: A new role for phospholipids in the regulation of cardiac calcium cycling?

The 52-amino acid transmembrane protein phospholamban (PLB) regulates calcium cycling in cardiac cells by forming a complex with the sarco(endo)plasmic reticulum calcium ATPase (SERCA) and reversibly diminishing the rate of calcium uptake by the sarcoplasmic reticulum. The N-terminal cytoplasmic domain of PLB interacts with the cytoplasmic domain of SERCA, but, in the absence of the enzyme, can also associate with the surface of anionic phospholipid membranes. This work investigates whether the cytoplasmic domain of PLB can also associate with membrane surfaces in the presence of SERCA, and whether such interactions could influence the regulation of the enzyme. It is shown using solid-state NMR and isothermal titration calorimetry (ITC) that an N-terminally acetylated peptide representing the first 23 N-terminal amino acids of PLB (PLB_1-23) interacts with membranes composed of zwitterionic phosphatidylcholine (PC) and anionic phosphatidylglycerol (PG) lipids in the absence and presence of SERCA. Functional measurements of SERCA in sarcoplasmic reticulum (SR) vesicles, planar SR membranes and reconstituted into PC/PG membranes indicate that PLB_1-23 lowers the maximal rate of ATP hydrolysis by acting at the cytoplasmic face of the enzyme. A small, but statistically significant, reduction in the inhibitory effect of the peptide is observed for SERCA reconstituted into PC/PG membranes compared to SERCA in membranes of PC alone. It is suggested that interactions between the cytoplasmic domain of PLB and negatively charged phospholipids might play a role in moderating the regulation of SERCA, with implications for cardiac muscle contractility.     

Effects of lipid composition on membrane permeabilization by sticholysin I and II, two cytolysins of the sea anemone Stichodactyla helianthus

Sticholysin I and II (St I and St II), two basic cytolysins purified from the Caribbean sea anemone Stichodactyla helianthus, efficiently permeabilize lipid vesicles by forming pores in their membranes. A general characteristic of these toxins is their preference for membranes containing sphingomyelin (SM). As a consequence, vesicles formed by equimolar mixtures of SM with phosphatidylcholine (PC) are very good targets for St I and II. To better characterize the lipid dependence of the cytolysin-membrane interaction, we have now evaluated the effect of including different lipids in the composition of the vesicles. We observed that at low doses of either St I or St II vesicles composed of SM and phosphatidic acid (PA) were permeabilized faster and to a higher extent than vesicles of PC and SM. As in the case of PC/SM mixtures, permeabilization was optimal when the molar ratio of PA/SM was ~1. The preference for membranes containing PA was confirmed by inhibition experiments in which the hemolytic activity of St I was diminished by pre-incubation with vesicles of different composition. The inclusion of even small proportions of PA into PC/SM LUVs led to a marked increase in calcein release caused by both St I and St II, reaching maximal effect at ~5 mol % of PA. Inclusion of other negatively charged lipids (phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI), or cardiolipin (CL)), all at 5 mol %, also elicited an increase in calcein release, the potency being in the order CL approximately PA > PG approximately PI approximately PS. However, some boosting effect was also obtained, including the zwitterionic lipid phosphatidylethanolamine (PE) or even, albeit to a lesser extent, the positively charged lipid stearylamine (SA). This indicated that the effect was not mediated by electrostatic interactions between the cytolysin and the negative surface of the vesicles. In fact, increasing the ionic strength of the medium had only a small inhibitory effect on the interaction, but this was actually larger with uncharged vesicles than with negatively charged vesicles. A study of the fluidity of the different vesicles, probed by the environment-sensitive fluorescent dye diphenylhexatriene (DPH), showed that toxin activity was also not correlated to the average membrane fluidity. It is suggested that the insertion of the toxin channel could imply the formation in the bilayer of a nonlamellar structure, a toroidal lipid pore. In this case, the presence of lipids favoring a nonlamellar phase, in particular PA and CL, strong inducers of negative curvature in the bilayer, could help in the formation of the pore. This possibility is confirmed by the fact that the formation of toxin pores strongly promotes the rate of transbilayer movement of lipid molecules, which indicates local disruption of the lamellar structure.     

'Detergent-like' permeabilization of anionic lipid vesicles by melittin

Melittin (MLT), the 26-residue toxic peptide from the European honeybee Apis mellifera, is widely used for studying the principles of membrane permeabilization by antimicrobial and other host-defense peptides. A striking property of MLT is that its ability to permeabilize zwitterionic phospholipid vesicles is dramatically reduced upon the addition of anionic lipids. Because the mechanism of permeabilization may be fundamentally different for the two types of lipids, we examined MLT-induced release of entrapped fluorescent dextran markers of two different molecular masses (4 and 50 kDa) from anionic palmitoyloleoylphosphatidylglycerol (POPG) vesicles. Unlike release from palmitoyloleoylphosphatidylcholine (POPC) vesicles, which is highly selective for the 4 kDa marker, implying release through pores of about 25 A diameter [Ladokhin et al., Biophys. J. 72 (1997) 1762], release from POPG vesicles was found to be non-selective, i.e., 'detergent-like'. Oriented circular dichroism measurements of MLT in oriented POPG and POPC multilayers disclosed that alpha-helical MLT can be induced to adopt a transbilayer orientation in POPC multilayers, but not in POPG multilayers. The apparent inhibition of MLT permeabilization by anionic membranes may thus be due to suppression of translocation ability.