Interaction of the eukaryotic pore-forming cytolysin equinatoxin II with model membranes: 19F NMR studies

Sea anemones produce a family of 18-20 kDa proteins, the actinoporins, which lyse cells by forming pores in cell membranes. Sphingomyelin plays an important role in their lytic activity, with membranes lacking this lipid being largely refractory to these toxins. As a means of characterising membrane binding by the actinoporin equinatoxin II (EqTII), we have used 19F NMR to probe the environment of Trp residues in the presence of micelles and bicelles. Trp was chosen as previous data from mutational studies and truncated analogues had identified the N-terminal helix of EqTII and the surface aromatic cluster including tryptophan residues 112 and 116 as being important for membrane interactions. The five tryptophan residues were replaced with 5-fluorotryptophan and assigned by site-directed mutagenesis. The 19F resonance of W112 was most affected in the presence of phospholipid micelles or bicelles, followed by W116, with further change induced by the addition of sphingomyelin. Although binding to phosphatidylcholine is not sufficient to enable pore formation in bilayer membranes, this interaction had a greater effect on the tryptophan residues in our studies than the subsequent interaction with sphingomyelin. Furthermore, sphingomyelin had a direct effect on EqTII in both model membranes, so its role in EqTII pore formation involves more than simply an indirect effect mediated via bulk lipid properties. The lack of change in chemical shift for W149 even in the presence of sphingomyelin indicates that, at least in the model membranes studied here, interaction with sphingomyelin was not sufficient to trigger dissociation of the N-terminal helix from the beta-sandwich, which forms the bulk of the protein.     

Membrane Damage by an α-Helical Pore-forming Protein,Equinatoxin II,Proceeds through a Succession of Ordered Steps

Actinoporin equinatoxin II (EqtII) is an archetypal example of α-helical pore-forming toxins that porate cellular membranes by the use of α-helices. Previous studies proposed several steps in the pore formation: binding of monomeric protein onto the membrane, followed by oligomerization and insertion of the N-terminal α-helix into the lipid bilayer. We studied these separate steps with an EqtII triple cysteine mutant. The mutant was engineered to monitor the insertion of the N terminus into the lipid bilayer by labeling Cys-18 with a fluorescence probe and at the same time to control the flexibility of the N-terminal region by the disulfide bond formed between cysteines introduced at positions 8 and 69. The insertion of the N terminus into the membrane proceeded shortly after the toxin binding and was followed by oligomerization. The oxidized, non-lytic, form of the mutant was still able to bind to membranes and oligomerize at the same level as the wild-type or the reduced form. However, the kinetics of the N-terminal helix insertion, the release of calcein from erythrocyte ghosts, and hemolysis of erythrocytes was much slower when membrane-bound oxidized mutant was reduced by the addition of the reductant. Results show that the N-terminal region needs to be inserted in the lipid membrane before the oligomerization into the final pore and imply that there is no need for a stable prepore formation. This is different from β-pore-forming toxins that often form β-barrel pores via a stable prepore complex.     

Molecular determinants of sphingomyelin specificity of a eukaryotic pore-forming toxin

Sphingomyelin (SM) is abundant in the outer leaflet of the cell plasma membrane, with the ability to concentrate in so-called lipid rafts. These specialized cholesterol-rich microdomains not only are associated with many physiological processes but also are exploited as cell entry points by pathogens and protein toxins. SM binding is thus a widespread and important biochemical function, and here we reveal the molecular basis of SM recognition by the membrane-binding eukaryotic cytolysin equinatoxin II (EqtII). The presence of SM in membranes drastically improves the binding and permeabilizing activity of EqtII. Direct binding assays showed that EqtII specifically binds SM, but not other lipids and, curiously, not even phosphatidylcholine, which presents the same phosphorylcholine headgroup. Analysis of the EqtII interfacial binding site predicts that electrostatic interactions do not play an important role in the membrane interaction and that the two most important residues for sphingomyelin recognition are Trp(112) and Tyr(113) exposed on a large loop. Experiments using site-directed mutagenesis, surface plasmon resonance, lipid monolayer, and liposome permeabilization assays clearly showed that the discrimination between sphingomyelin and phosphatidylcholine occurs in the region directly below the phosphorylcholine headgroup. Because the characteristic features of SM chemistry lie in this subinterfacial region, the recognition mechanism may be generic for all SM-specific proteins.     

Structure and activity of the N-terminal region of the eukaryotic cytolysin equinatoxin II

The actinoporins are a family of proteins from sea anemones that lyse cells by forming pores in cell membranes. Sphingomyelin plays an important role in their lytic activity, with membranes lacking this lipid being resistant to these toxins. Pore formation by the actinoporin equinatoxin II (EqTII) proceeds by membrane binding via a surface rich in aromatic residues, followed by translocation of the N-terminal region to the membrane and, finally, across the bilayer to form a functional pore. A key feature of this mechanism is the ability of the N-terminal region to form a stable, bilayer-spanning helix in the membrane, which in turn requires dissociation of the N-terminus from the bulk of the protein and significant extension of the N-terminal helix of native EqTII. In this study the structures of three peptides corresponding to residues 11-29, 11-32, and 1-32, respectively, of EqTII have been investigated by high-resolution nuclear magnetic resonance and Fourier transform infrared spectroscopy. The 32-residue peptide lacks ordered secondary structure in water, but residues 6-28 form a helix in dodecylphosphocholine micelles. Although this helix is long enough to span a bilayer membrane, this peptide and the shorter analogues display limited permeabilizing activity in large unilamellar vesicles and very weak hemolytic activity in human red blood cells. Thus, while the N-terminal region has the structural features required for this unusual mechanism of pore formation, the lack of activity of the isolated N-terminus shows that the bulk of the protein is essential for efficient pore formation by facilitating initial membrane binding, interacting with sphingomyelin, or stabilizing the oligomeric pore.     

The behavior of sea anemone actinoporins at the water-membrane interface

Actinoporins constitute a group of small and basic α-pore forming toxins produced by sea anemones. They display high sequence identity and appear as multigene families. They show a singular behaviour at the water-membrane interface: In aqueous solution, actinoporins remain stably folded but, upon interaction with lipid bilayers, become integral membrane structures. These membranes contain sphingomyelin, display phase coexistence, or both. The water soluble structures of the actinoporins equinatoxin II (EqtII) and sticholysin II (StnII) are known in detail. The crystalline structure of a fragaceatoxin C (FraC) nonamer has been also determined. The three proteins fold as a β-sandwich motif flanked by two α-helices, one of them at the N-terminal end. Four regions seem to be especially important: A cluster of aromatic residues, a phosphocholine binding site, an array of basic amino acids, and the N-terminal α-helix. Initial binding of the soluble monomers to the membrane is accomplished by the cluster of aromatic amino acids, the array of basic residues, and the phosphocholine binding site. Then, the N-terminal α-helix detaches from the β-sandwich, extends, and lies parallel to the membrane. Simultaneously, oligomerization occurs. Finally, the extended N-terminal α-helix penetrates the membrane to build a toroidal pore. This model has been however recently challenged by the cryo-EM reconstruction of FraC bound to phospholipid vesicles. Actinoporins structural fold appears across all eukaryotic kingdoms in other functionally unrelated proteins. Many of these proteins neither bind to lipid membranes nor induce cell lysis. Finally, studies focusing on the therapeutic potential of actinoporins also abound.     

Effect of lipid on the conformation of the N-terminal region of equinatoxin II: a synchrotron radiation circular dichroism spectroscopic study

Equinatoxin II (EqtII) is a protein toxin that lyses both red blood cells and artificial membranes. Lysis is dependent on the lipid composition, with small unilamellar vesicles (SUVs) of dimyristoylphosphatidylcholine (DMPC) and sphingomyelin (SM) (1:1 molar) being lysed more readily than those of phosphatidylcholine alone. Removing the N-terminus of EqtII prevents pore formation, but does not prevent membrane binding. A peptide corresponding to residues 1–32 of EqtII was found using NMR to adopt a helical structure in micelles. To further understand the structural changes that accompany membrane insertion, synchrotron radiation circular dichroism spectra of the N-terminal peptide in a range of model membranes have been analysed. The peptide structure was examined in water, dodecylphosphocholine (DPC) and DPC:SM (5:1) micelles, and SUVs composed of dioleoylphosphatidylcholine (DOPC) or DMPC, together with SM and cholesterol (Chol). The peptide adopted different conformations in different lipids. Although the presence of SM did not affect the conformation in micelles, inclusion of SM in the bilayer-forming lipid increased the helicity of the peptide. This effect was abolished when Chol was added in DOPC but not in DMPC, which may relate to liquid ordered versus disordered phase properties of the lipid. SM may act as a promoter of membrane organisation necessary for membrane lysis by EqtII.     

The effects of lipids on the structure of the eukaryotic cytolysin equinatoxin II: a synchrotron radiation circular dichroism spectroscopic study

Synchrotron radiation circular dichroism (SRCD) spectroscopy studies of the eukaryotic pore-forming protein equinatoxin II (EqtII) were carried out in solution and in the presence of micelles or small unilamellar vesicles (SUV) of different lipid composition. The SRCD structural data was correlated with calcein leakage from SUV and with partitioning of EqtII to liposomes, and micelles, according to haemolysis assays. The structure of EqtII in water and dodecylphosphocholine micelles as determined by SRCD was similar to the values calculated from crystal and solution structures of the protein, and no changes were observed with the addition of sphingomyelin (SM). SM is required to trigger pore formation in biological and model membranes, but our results suggest that SM alone is not sufficient to trigger dissociation of the N-terminal helix and further structural rearrangements required to produce a pore. Significant changes in conformation of EqtII were detected with unsaturated phospholipid (DOPC) vesicles when SM was added, but not with saturated phospholipids (DMPC), which suggests that not only is membrane curvature important, but also the fluidity of the bilayer. The SRCD data indicated that the EqtII structure in the presence of DOPC:SM SUV represents the 'bound' state and the 'free' state is represented by spectra for DOPC or DOPC:Chol vesicles, which correlates with the high lytic activity for SUV of DOPC:SM. The SRCD results provide insight into the lipid requirements for structural rearrangements associated with EqtII toxicity and lysis.     

Characterization of the Lipid-Binding Site of Equinatoxin II by NMR and Molecular Dynamics Simulation

Equinatoxin II (EqtII) is a soluble, 20 kDa pore-forming protein toxin isolated from the sea anemone Actinia equina. Although pore formation has long been known to occur in distinct stages, including monomeric attachment to phospholipid membranes followed by detachment of the N-terminal helical domain and oligomerization into the final pore assembly, atomistic-level detail of the protein-lipid interactions underlying these events remains elusive. Using high-resolution solution state NMR of uniformly-¹⁵N-labeled EqtII at the critical micelle concentration of dodecylphosphocholine, we have mapped the lipid-binding site through chemical shift perturbations. Subsequent docking of an EqtII monomer onto a dodecylphosphocholine micelle, followed by 400 ns of all-atom molecular dynamics simulation, saw several high-occupancy lipid-binding pockets stabilized by cation-π, hydrogen bonding, and hydrophobic interactions; and stabilization of the loop housing the conserved arginine-glycine-aspartate motif. Additional simulation of EqtII with an N-acetyl sphingomyelin micelle, for which high-resolution NMR data cannot be obtained due to aggregate formation, revealed that sphingomyelin specificity might occur via hydrogen bonding to the 3-OH and 2-NH groups unique to the ceramide backbone by side chains of D109 and Y113; and main chains of P81 and W112. Furthermore, a binding pocket formed by K30, K77, and P81, proximate to the hinge region of the N-terminal helix, was identified and may be implicated in triggering pore formation.     

Crystal structure of the soluble form of equinatoxin II, a pore-forming toxin from the sea anemone Actinia equina

BACKGROUND: Membrane pore-forming toxins have a remarkable property: they adopt a stable soluble form structure, which, when in contact with a membrane, undergoes a series of transformations, leading to an active, membrane-bound form. In contrast to bacterial toxins, no structure of a pore-forming toxin from an eukaryotic organism has been determined so far, an indication that structural studies of equinatoxin II (EqtII) may unravel a novel mechanism. RESULTS: The crystal structure of the soluble form of EqtII from the sea anemone Actinia equina has been determined at 1.9 A resolution. EqtII is shown to be a single-domain protein based on a 12 strand beta sandwich fold with a hydrophobic core and a pair of alpha helices, each of which is associated with the face of a beta sheet. CONCLUSIONS: The structure of the 30 N-terminal residues is the largest segment that can adopt a different structure without disrupting the fold of the beta sandwich core. This segment includes a three-turn alpha helix that lies on the surface of a beta sheet and ends in a stretch of three positively charged residues, Lys-30, Arg-31, and Lys-32. On the basis of gathered data, it is suggested that this segment forms the membrane pore, whereas the beta sandwich structure remains unaltered and attaches to a membrane as do other structurally related extrinsic membrane proteins or their domains. The use of a structural data site-directed mutagenesis study should reveal the residues involved in membrane pore formation.     

The effects of lipids on the structure of the eukaryotic cytolysin equinatoxin II: A synchrotron radiation circular dichroism spectroscopic study

Synchrotron radiation circular dichroism (SRCD) spectroscopy studies of the eukaryotic pore-forming protein equinatoxin II (EqtII) were carried out in solution and in the presence of micelles or small unilamellar vesicles (SUV) of different lipid composition. The SRCD structural data was correlated with calcein leakage from SUV and with partitioning of EqtII to liposomes, and micelles, according to haemolysis assays. The structure of EqtII in water and dodecylphosphocholine micelles as determined by SRCD was similar to the values calculated from crystal and solution structures of the protein, and no changes were observed with the addition of sphingomyelin (SM). SM is required to trigger pore formation in biological and model membranes, but our results suggest that SM alone is not sufficient to trigger dissociation of the N-terminal helix and further structural rearrangements required to produce a pore. Significant changes in conformation of EqtII were detected with unsaturated phospholipid (DOPC) vesicles when SM was added, but not with saturated phospholipids (DMPC), which suggests that not only is membrane curvature important, but also the fluidity of the bilayer. The SRCD data indicated that the EqtII structure in the presence of DOPC:SM SUV represents the ‘bound’ state and the ‘free’ state is represented by spectra for DOPC or DOPC:Chol vesicles, which correlates with the high lytic activity for SUV of DOPC:SM. The SRCD results provide insight into the lipid requirements for structural rearrangements associated with EqtII toxicity and lysis.     

Effect of lipid composition on the "membrane response" induced by a fusion peptide

To understand the initial stages of membrane destabilization induced by viral proteins, the factors important for binding of fusion peptides to cell membranes must be identified. In this study, effects of lipid composition on the mode of peptides' binding to membranes are explored via molecular dynamics (MD) simulations of the peptide E5, a water-soluble analogue of influenza hemagglutinin fusion peptide, in two full-atom hydrated lipid bilayers composed of dimyristoyl- and dipalmitoylphosphatidylcholine (DMPC and DPPC, respectively). The results show that, although the peptide has a common folding motif in both systems, it possesses different modes of binding. The peptide inserts obliquely into the DMPC membrane mainly with its N-terminal alpha helix, while in DPPC, the helix lies on the lipid/water interface, almost parallel to the membrane surface. The peptide seriously affects structural and dynamical parameters of surrounding lipids. Thus, it induces local thinning of both bilayers and disordering of acyl chains of lipids in close proximity to the binding site. The "membrane response" significantly depends upon lipid composition: distortions of DMPC bilayer are more pronounced than those in DPPC. Implications of the observed effects to molecular events on initial stages of membrane destabilization induced by fusion peptides are discussed.     

Imaging the Lipid-Phase-Dependent Pore Formation of Equinatoxin II in Droplet Interface Bilayers

Using phase-separated droplet interface bilayers, we observe membrane binding and pore formation of a eukaryotic cytolysin, Equinatoxin II (EqtII). EqtII activity is known to depend on the presence of sphingomyelin in the target membrane and is enhanced by lipid phase separation. By imaging the ionic flux through individual pores in vitro, we observe that EqtII pores form predominantly within the liquid-disordered phase. We observe preferential binding of labeled EqtII at liquid-ordered/liquid-disordered domain boundaries before it accumulates in the liquid-disordered phase.     

Effects of the eukaryotic pore-forming cytolysin Equinatoxin II on lipid membranes and the role of sphingomyelin

Equinatoxin II (EqtII), a protein toxin from the sea anemone Actinia equina, readily creates pores in sphingomyelin-containing lipid membranes. The perturbation by EqtII of model lipid membranes composed of dimyristoylphosphatidycholine and sphingomyelin (10 mol %) was investigated using wideline phosphorus-31 and deuterium NMR. The preferential interaction between EqtII (0.1 and 0.4 mol %) and the individual bilayer lipids was studied by (31)P magic angle spinning NMR, and toxin-induced changes in bilayer morphology were examined by freeze-fracture electron microscopy. Both NMR and EM showed the formation of an additional lipid phase in sphingomyelin-containing mixed lipid multilamellar suspensions with 0.4 mol % EqtII. The new toxin-induced phase consisted of small unilamellar vesicles 20-40 nm in diameter. Deuterium NMR showed that the new lipid phase contains both dimyristoylphosphatidycholine and sphingomyelin. Solid-state (31)P NMR showed an increase in spin-lattice and a decrease in spin-spin relaxation times in mixed-lipid model membranes in the presence of EqtII, consistent with an increase in the intensity of low frequency motions. The (2)H and (31)P spectral intensity distributions confirmed a change in lipid mobility and showed the creation of an isotropic lipid phase, which was identified as the small vesicle structures visible by electron microscopy in the EqtII-lipid suspensions. The toxin appears to enhance slow motions in the membrane lipids and destabilize the membrane. This effect was greatly enhanced in sphingomyelin-containing mixed lipid membranes compared with pure phosphatidylcholine bilayers, suggesting a preferential interaction between the toxin and bilayer sphingomyelin.     

Imaging the Lipid-Phase-Dependent Pore Formation of Equinatoxin II in Droplet Interface Bilayers

Using phase-separated droplet interface bilayers, we observe membrane binding and pore formation of a eukaryotic cytolysin, Equinatoxin II (EqtII). EqtII activity is known to depend on the presence of sphingomyelin in the target membrane and is enhanced by lipid phase separation. By imaging the ionic flux through individual pores in vitro, we observe that EqtII pores form predominantly within the liquid-disordered phase. We observe preferential binding of labeled EqtII at liquid-ordered/liquid-disordered domain boundaries before it accumulates in the liquid-disordered phase.     

Cell-cell membrane fusion induced by p15 fusion-associated small transmembrane (FAST) protein requires a novel fusion peptide motif containing a myristoylated polyproline type II helix

The p15 fusion-associated small transmembrane (FAST) protein is a nonstructural viral protein that induces cell-cell fusion and syncytium formation. The exceptionally small, myristoylated N-terminal ectodomain of p15 lacks any of the defining features of a typical viral fusion protein. NMR and CD spectroscopy indicate this small fusion module comprises a left-handed polyproline type II (PPII) helix flanked by small, unstructured N and C termini. Individual prolines in the 6-residue proline-rich motif are highly tolerant of alanine substitutions, but multiple substitutions that disrupt the PPII helix eliminate cell-cell fusion activity. A synthetic p15 ectodomain peptide induces lipid mixing between liposomes, but with unusual kinetics that involve a long lag phase before the onset of rapid lipid mixing, and the length of the lag phase correlates with the kinetics of peptide-induced liposome aggregation. Lipid mixing, liposome aggregation, and stable peptide-membrane interactions are all dependent on both the N-terminal myristate and the presence of the PPII helix. We present a model for the mechanism of action of this novel viral fusion peptide, whereby the N-terminal myristate mediates initial, reversible peptide-membrane binding that is stabilized by subsequent amino acid-membrane interactions. These interactions induce a biphasic membrane fusion reaction, with peptide-induced liposome aggregation representing a distinct, rate-limiting event that precedes membrane merger. Although the prolines in the proline-rich motif do not directly interact with membranes, the PPII helix may function to force solvent exposure of hydrophobic amino acid side chains in the regions flanking the helix to promote membrane binding, apposition, and fusion.     

Solution structure and interaction of cupiennin 1a, a spider venom peptide, with phospholipid bilayers

The solution structure of cupiennin 1a, a 35 residue, basic antibacterial peptide isolated from the venom of the spider Cupiennius salei, has been determined by nuclear magnetic resonance (NMR) spectroscopy. The peptide was found to adopt a helix-hinge-helix structure in a membrane mimicking solvent. The hinge may play a role in allowing the amphipathic N-terminal helix and polar C-terminal helix to orient independently upon membrane binding, in order to achieve maximal antibacterial efficacy. Solid-state 31P and 2H NMR was used to further study the effects of cupiennin 1a on the dynamic properties of lipid membranes, using zwitterionic chain deuterated dimyristoylphosphatidylcholine (d54-DMPC) and anionic dimyristoylphosphatidylglycerol (DMPG) multilamellar vesicles. In d54-DMPC alone, cupiennin 1a caused a decrease in the 31P chemical shift anisotropy, indicating some interaction with the lipid head groups, and a decrease in order over the entire acyl chain. In contrast, for the mixed (d54-DMPC/DMPG) lipid system cupiennin 1a appeared to induce lateral separation of the two lipids as evidenced by the 31P spectra, in which the peptide preferentially interacted with DMPG. Little effect was observed on the deuterated acyl chain order parameters in the d54-DMPC/DMPG model membranes. Furthermore, 31P NMR relaxation measurements confirmed a differential effect on the lipid motions depending upon the membrane composition. Therefore, subtle differences are likely in the mechanism by which cupiennin 1a causes membrane lysis in either prokaryotic or eukaryotic cells, and may explain the specific spectrum of activity.     

Solution structures and model membrane interactions of lactoferrampin, an antimicrobial peptide derived from bovine lactoferrin

Bovine lactoferrampin (LFampinB) has been identified as a novel antimicrobial peptide, which is derived from the N-terminal lobe of bovine lactoferrin. In this study, the solution structure of LFampinB bound to negatively charged sodium dodecyl sulphate micelles and zwitterionic dodecyl phosphocholine micelles was determined using 2-dimensional nuclear magnetic resonance (NMR) spectroscopy. The interaction between LFampinB and multilamellar phospholipid vesicles, containing choline and glycerol head groups, was examined using differential scanning calorimetry (DSC). In addition, the interaction between the N-terminal tryptophan residue and model membranes of varying composition was analyzed by fluorescence spectroscopy. LFampinB adopts an amphipathic alpha-helical conformation across the first 11 residues of the peptide but remains relatively unstructured at the C-terminus. The hydrophobic surface of the amphipathic helix is bordered by the side chains of Trp1 and Phe11, and is seen in both micelle-bound structures. The fluorescence results suggest that Trp1 inserts into the membrane at the lipid/water interface. The phenyl side chain of Phe11 is oriented in the same direction as the indole ring of Trp1, allowing these two residues to serve as anchors for the lipid bilayer. The DSC results also indicate that LFampinB interacts with glycerol head groups in multilamellar vesicles but has little effect on acyl chain packing. Our results support a two step model of antimicrobial activity where the initial attraction of LFampinB is mediated by the cluster of positive charges on the C-terminus followed by the formation of the N-terminal helix which binds to the surface of the bacterial lipid bilayer.     

Solution structures and model membrane interactions of lactoferrampin, an antimicrobial peptide derived from bovine lactoferrin

Bovine lactoferrampin (LFampinB) has been identified as a novel antimicrobial peptide, which is derived from the N-terminal lobe of bovine lactoferrin. In this study, the solution structure of LFampinB bound to negatively charged sodium dodecyl sulphate micelles and zwitterionic dodecyl phosphocholine micelles was determined using 2-dimensional nuclear magnetic resonance (NMR) spectroscopy. The interaction between LFampinB and multilamellar phospholipid vesicles, containing choline and glycerol head groups, was examined using differential scanning calorimetry (DSC). In addition, the interaction between the N-terminal tryptophan residue and model membranes of varying composition was analyzed by fluorescence spectroscopy. LFampinB adopts an amphipathic alpha-helical conformation across the first 11 residues of the peptide but remains relatively unstructured at the C-terminus. The hydrophobic surface of the amphipathic helix is bordered by the side chains of Trp1 and Phe11, and is seen in both micelle-bound structures. The fluorescence results suggest that Trp1 inserts into the membrane at the lipid/water interface. The phenyl side chain of Phe11 is oriented in the same direction as the indole ring of Trp1, allowing these two residues to serve as anchors for the lipid bilayer. The DSC results also indicate that LFampinB interacts with glycerol head groups in multilamellar vesicles but has little effect on acyl chain packing. Our results support a two step model of antimicrobial activity where the initial attraction of LFampinB is mediated by the cluster of positive charges on the C-terminus followed by the formation of the N-terminal helix which binds to the surface of the bacterial lipid bilayer.     

Weak Glycolipid Binding of a Microdomain-Tracer Peptide Correlates with Aggregation and Slow Diffusion on Cell Membranes

Organized assembly or aggregation of sphingolipid-binding ligands, such as certain toxins and pathogens, has been suggested to increase binding affinity of the ligand to the cell membrane and cause membrane reorganization or distortion. Here we show that the diffusion behavior of the fluorescently tagged sphingolipid-interacting peptide probe SBD (Sphingolipid Binding Domain) is altered by modifications in the construction of the peptide sequence that both result in a reduction in binding to ganglioside-containing supported lipid membranes, and at the same time increase aggregation on the cell plasma membrane, but that do not change relative amounts of secondary structural features. We tested the effects of modifying the overall charge and construction of the SBD probe on its binding and diffusion behavior, by Surface Plasmon Resonance (SPR; Biacore) analysis on lipid surfaces, and by Fluorescence Correlation Spectroscopy (FCS) on live cells, respectively. SBD binds preferentially to membranes containing the highly sialylated gangliosides GT1b and GD1a. However, simple charge interactions of the peptide with the negative ganglioside do not appear to be a critical determinant of binding. Rather, an aggregation-suppressing amino acid composition and linker between the fluorophore and the peptide are required for optimum binding of the SBD to ganglioside-containing supported lipid bilayer surfaces, as well as for interaction with the membrane. Interestingly, the strength of interactions with ganglioside-containing artificial membranes is mirrored in the diffusion behavior by FCS on cell membranes, with stronger binders displaying similar characteristic diffusion profiles. Our findings indicate that for aggregation-prone peptides, aggregation occurs upon contact with the cell membrane, and rather than giving a stronger interaction with the membrane, aggregation is accompanied by weaker binding and complex diffusion profiles indicative of heterogeneous diffusion behavior in the probe population.     

The dispensability and requirement of SecA N-terminal aminoacyl residues for complementation,membrane binding,lipid-specific domains and channel activities

SecA is an essential multifunctional protein for the translocation of proteins across bacterial membranes. Though SecA is known to function in the membrane, the detailed mechanism for this process remains unclear. In this study we constructed a series of SecA N-terminal deletions and identified two specific domains crucial for initial SecA/membrane interactions. The first small helix, the linker and part of the second helix (Δ2-22) were found to be dispensable for SecA activity in complementing the growth of a SecA ts mutant. However, deletions of N-terminal aminoacyl residues 23–25 resulted in severe progressive retardation of growth. Moreover, a decrease of SecA activity caused by N-terminal deletions correlated to the loss of SecA membrane binding, formation of lipid-specific domains and channel activity. All together, the results indicate that the N-terminal aminoacyl residues 23–25 play a critical role for SecA binding to membranes and that the N-terminal limit of SecA for activity is at the 25th amino acid.