The role played by lipids unsaturation upon the membrane interaction of the Helicobacter pylori HP(2–20) antimicrobial peptide analogue HPA3

The HPA3 peptide is an analogue of the linear antimicrobial peptide, HP(2–20), isolated from the N-terminal region of the Helicobacter pylori ribosomal protein, able to interact with zwitterionic lipid membranes and generate pores. Herein we focused on the importance of the degree of unsaturation of lipid acyl chains on HPA3 peptide-membrane interactions. Electrophysiology experiments carried out in reconstituted lipid membranes formed from phosphatidylcholines with one (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine − POPC) and two monounsaturated acyl chains (1,2-dioleoyl-sn-glycero-3-phosphocholine − DOPC) demonstrate that the lesser degree of the packing density of membrane lipids encountered in DOPC-based planar membranes greatly enhances the electric activity of pores created by the HPA3 peptide. Data derived from fluorescence spectroscopy experiments demonstrate that upon interaction with the bilayer, the HPA3 peptide translocates to the trans-side of the membrane. From the same experiments, we demonstrate that in the case of DOPC-based planar membranes, the net amount of HPA3 peptide which passes across the membrane and re-dissolves in the trans solution is almost 22% greater than POPC-based membranes. Such data further emphasize the modulatory role played by lipid acyl chain in determining antimicrobial peptides-lipids interactions, and demonstrate that small differences in unsaturation degree can impose a sizeable influence on HPA3 peptide activity.     

Interaction of hagfish cathelicidin antimicrobial peptides with model lipid membranes

Hagfish intestinal antimicrobial peptides (HFIAPs) are a family of polycationic peptides exhibiting potent, broad-spectrum bactericidal activity. In an attempt to unravel the mechanism of action of HFIAPs, we have studied their interaction with model membranes. Synthetic HFIAPs selectively bound to liposomes mimicking bacterial membranes, and caused the release of vesicle-encapsulated fluorescent markers in a size-dependent manner. In planar lipid bilayer membranes, HFIAPs induced erratic current fluctuations and reduced membrane line tension according to a general theory for lipidic pores, suggesting that HFIAP pores contain lipid molecules. Consistent with this notion, lipid transbilayer redistribution accompanied HFIAP pore formation, and membrane monolayer curvature regulated HFIAP pore formation. Based on these studies, we propose that HFIAPs kill target cells, at least in part, by interacting with their plasma membrane to induce formation of lipid-containing pores. Such a membrane-permeabilizing function appears to be an evolutionarily conserved host-defense mechanism of antimicrobial peptides.     

Defect formation of lytic peptides in lipid membranes and their influence on the thermodynamic properties of the pore environment

We present an experimental study of the pore formation processes of small amphipathic peptides in model phosphocholine lipid membranes. We used atomic force microscopy to characterize the spatial organization and structure of alamethicin- and melittin-induced defects in lipid bilayer membranes and the influence of the peptide on local membrane properties. Alamethicin induced holes in gel DPPC membranes were directly visualized at different peptide concentrations. We found that the thermodynamic state of lipids in gel membranes can be influenced by the presence of alamethicin such that nanoscopic domains of fluid lipids form close to the peptide pores, and that the elastic constants of the membrane are altered in their vicinity. Melittin-induced holes were visualized in DPPC and DLPC membranes at room temperature in order to study the influence of the membrane state on the peptide induced hole formation. Also differential scanning calorimetry was used to investigate the effect of alamethicin on the lipid membrane phase behaviour.     

Defect formation of lytic peptides in lipid membranes and their influence on the thermodynamic properties of the pore environment

We present an experimental study of the pore formation processes of small amphipathic peptides in model phosphocholine lipid membranes. We used atomic force microscopy to characterize the spatial organization and structure of alamethicin- and melittin-induced defects in lipid bilayer membranes and the influence of the peptide on local membrane properties. Alamethicin induced holes in gel DPPC membranes were directly visualized at different peptide concentrations. We found that the thermodynamic state of lipids in gel membranes can be influenced by the presence of alamethicin such that nanoscopic domains of fluid lipids form close to the peptide pores, and that the elastic constants of the membrane are altered in their vicinity. Melittin-induced holes were visualized in DPPC and DLPC membranes at room temperature in order to study the influence of the membrane state on the peptide induced hole formation. Also differential scanning calorimetry was used to investigate the effect of alamethicin on the lipid membrane phase behaviour.     

Transmembrane pores formed by human antimicrobial peptide LL-37

Human LL-37 is a multifunctional cathelicidin peptide that has shown a wide spectrum of antimicrobial activity by permeabilizing microbial membranes similar to other antimicrobial peptides; however, its molecular mechanism has not been clarified. Two independent experiments revealed LL-37 bound to membranes in the α-helical form with the axis lying in the plane of membrane. This led to the conclusion that membrane permeabilization by LL-37 is a nonpore carpet-like mechanism of action. Here we report the detection of transmembrane pores induced by LL-37. The pore formation coincided with LL-37 helices aligning approximately normal to the plane of the membrane. We observed an unusual phenomenon of LL-37 embedded in stacked membranes, which are commonly used in peptide orientation studies. The membrane-bound LL-37 was found in the normal orientation only when the membrane spacing in the multilayers exceeded its fully hydrated value. This was achieved by swelling the stacked membranes with excessive water to a swollen state. The transmembrane pores were detected and investigated in swollen states by means of oriented circular dichroism, neutron in-plane scattering, and x-ray lamellar diffraction. The results are consistent with the effect of LL-37 on giant unilamellar vesicles. The detected pores had a water channel of radius 23–33 Å. The molecular mechanism of pore formation by LL-37 is consistent with the two-state model exhibited by magainin and other small pore-forming peptides. The discovery that peptide-membrane interactions in swollen states are different from those in less hydrated states may have implications for other large membrane-active peptides and proteins studied in stacked membranes.     

Pore formation by a Bax-derived peptide: effect on the line tension of the membrane probed by AFM

Bax is a critical regulator of physiological cell death that increases the permeability of the outer mitochondrial membrane and facilitates the release of the so-called apoptotic factors during apoptosis. The molecular mechanism of action is unknown, but it probably involves the formation of partially lipidic pores induced by Bax. To investigate the interaction of Bax with lipid membranes and the physical changes underlying the formation of Bax pores, we used an active peptide derived from helix 5 of this protein (Bax-alpha5) that is able to induce Bax-like pores in lipid bilayers. We report the decrease of line tension due to peptide binding both at the domain interface in phase-separated lipid bilayers and at the pore edge in atomic force microscopy film-rupture experiments. Such a decrease in line tension may be a general strategy of pore-forming peptides and proteins, as it affects the energetics of the pore and stabilizes the open state.     

Toroidal pores formed by antimicrobial peptides show significant disorder

A large variety of antimicrobial peptides have been shown to act, at least in vitro, by poration of the lipid membrane. The nanometre size of these pores, however, complicates their structural characterization by experimental techniques. Here we use molecular dynamics simulations, to study the interaction of a specific class of antimicrobial peptides, melittin, with a dipalmitoylphosphatidylcholine bilayer in atomic detail. We show that transmembrane pores spontaneously form above a critical peptide to lipid ratio. The lipid molecules bend inwards to form a toroidally shaped pore but with only one or two peptides lining the pore. This is in strong contrast to the traditional models of toroidal pores in which the peptides are assumed to adopt a transmembrane orientation. We find that peptide aggregation, either prior or after binding to the membrane surface, is a prerequisite to pore formation. The presence of a stable helical secondary structure of the peptide, however is not. Furthermore, results obtained with modified peptides point to the importance of electrostatic interactions in the poration process. Removing the charges of the basic amino-acid residues of melittin prevents pore formation. It was also found that in the absence of counter ions pores not only form more rapidly but lead to membrane rupture. The rupture process occurs via a novel recursive poration pathway, which we coin the Droste mechanism.     

Single-channel analysis of the conductance fluctuations induced in lipid bilayer membranes by complement proteins C5b-9

Summary Single-channel analysis of electrical fluctuations induced in planar bilayer membranes by the purified human complement proteins C5b6, C7, C8, and C9 have been analyzed. Reconstitution experiments with lipid bilayer membranes showed that the C5b-9 proteins formed pores only if all proteins were present at one side of the membrane. The complement pores had an average single-channel conductance of 3.1 nS at 0.15m KCl. The histogram of the complement pores suggested a substantial variation of the size of the single channel. The linear relationship between single-channel conductance at fixed ionic strength and the aqueous mobility of the ions in the bulk aqueous phase indicated that the ions move inside the complement pore in a manner similar to the way they move in the aqueous phase. The minimum diameter of the pores as judged from the conductance data is approximately 3 nm. The complement channels showed no apparent voltage control or regulation up to transmembrane potentials of 100 mV. At neutral pH the pore is three to four times more permeable for alkali ions than for chloride, which may be explained by the existence of fixed negatively charged groups in or near the pore. The significance of these observations to current molecular models of the membrane lesion formed by these cytolytic serum proteins is considered.     

The membrane insertion of helical antimicrobial peptides from the N-terminus of Helicobacter pylori ribosomal protein L1

The interaction of two helical antimicrobial peptides, HPA3 and HPA3P with planar supported lipid membranes was quantitatively analysed using two complementary optical biosensors. The peptides are analogues of Hp(2-20) derived from the N-terminus of Helicobacter pylori ribosomal protein L1 (RpL1). The binding of these two peptide analogues to zwitterionic dimyristoyl-phosphatidylcholine (DMPC) and negatively charged membranes composed of DMPC/dimyristoylphosphatidylglycerol (DMPG) (4:1) was determined using surface plasmon resonance (SPR) and dual polarisation interferometry (DPI). Using SPR analysis, it was shown that the proline substitution in HPA3P resulted in much lower binding for both zwitterionic and anionic membranes than HPA3. Structural changes in the planar DMPC and DMPC/DMPG (4:1) bilayers induced by the binding of both Hp(2-20) analogues were then resolved in real-time with DPI. The overall process of peptide-induced changes in membrane structure was analysed by the real-time changes in bound peptide mass as a function of bilayer birefringence. The insertion of both HPA3 and HPA3P into the supported lipid bilayers resulted in a decrease in birefringence with increasing amounts of bound peptide which reflects a decrease in the order of the bilayer. The binding of HPA3 to each membrane was associated with a higher level of bound peptide and greater membrane lipid disordering and a faster and higher degree of insertion into the membrane than HPA3P. Furthermore, the binding of both HPA3 and HPA3P to negatively charged DMPC/DMPG bilayers also leads to a greater disruption of the lipid ordering. These results demonstrate the geometrical changes in the membrane upon peptide insertion and the extent of membrane structural changes can be obtained quantitatively. Moreover, monitoring the effect of peptides on a structurally characterised bilayer has provided further insight into the role of membrane structure changes in the molecular basis of peptide selectivity and activity and may assist in defining the mode of antimicrobial action.     

Molecular mechanism of antimicrobial peptides: the origin of cooperativity

Based on very extensive studies on four peptides (alamethicin, melittin, magainin and protegrin), we propose a mechanism to explain the cooperativity exhibited by the activities of antimicrobial peptides, namely, a non-linear concentration dependence characterized by a threshold and a rapid rise to saturation as the concentration exceeds the threshold. We first review the structural basis of the mechanism. Experiments showed that peptide binding to lipid bilayers creates two distinct states depending on the bound-peptide to lipid ratio P/L. For P/L below a threshold P/L*, all of the peptide molecules are in the S state that has the following characteristics: (1) there are no pores in the membrane, (2) the axes of helical peptides are oriented parallel to the plane of membrane, and (3) the peptide causes membrane thinning in proportion to P/L. As P/L increases above P/L*, essentially all of the excessive peptide molecules occupy the I state that has the following characteristics: (1) transmembrane pores are detected in the membrane, (2) the axes of helical peptides are perpendicular to the plane of membrane, (3) the membrane thickness remains constant for P/L> or =P/L*. The free energy based on these two states agrees with the data quantitatively. The free energy also explains why lipids of positive curvature (lysoPC) facilitate and lipids of negative curvature (PE) inhibit pore formation.     

The aromatic domain of the coronavirus class I viral fusion protein induces membrane permeabilization: putative role during viral entry

Coronavirus (CoV) entry is mediated by the viral spike (S) glycoprotein, a class I viral fusion protein. During viral and target cell membrane fusion, the heptad repeat (HR) regions of the S2 subunit assume a trimer-of-hairpins structure, positioning the fusion peptide in close proximity to the C-terminal region of the ectodomain. The formation of this structure appears to drive apposition and subsequent fusion of viral and target cell membranes; however, the exact mechanism is unclear. Here, we characterize an aromatic amino acid rich region within the ectodomain of the S2 subunit that both partitions into lipid membranes and has the capacity to perturb lipid vesicle integrity. Circular dichroism analysis indicated that peptides analogous to the aromatic domains of the severe acute respiratory syndrome (SARS)-CoV, mouse hepatitis virus (MHV) and the human CoV OC43 S2 subunits, did not have a propensity for a defined secondary structure. These peptides strongly partitioned into lipid membranes and induced lipid vesicle permeabilization at peptide/lipid ratios of 1:100 in two independent leakage assays. Thus, partitioning of the peptides into the lipid interface is sufficient to disorganize membrane integrity. Our study of the S2 aromatic domain of three CoVs provides supportive evidence for a functional role of this region. We propose that, when aligned with the fusion peptide and transmembrane domains during membrane apposition, the aromatic domain of the CoV S protein functions to perturb the target cell membrane and provides a continuous track of hydrophobic surface, resulting in lipid-membrane fusion and subsequent viral nucleocapsid entry.     

Membrane Lipids Determine the Antibiotic Activity of the Lantibiotic Gallidermin

Lantibiotics, a group of lanthionine-containing peptides, display their antibiotic activity by combining different killing mechanisms within one molecule. The prototype lantibiotic nisin was shown to possess both inhibition of peptidoglycan synthesis and pore formation in bacterial membranes by interacting with lipid II. Gallidermin, which shares the lipid II binding motif with nisin but has a shorter molecular length, differed from nisin in pore formation in several strains of bacteria. To simulate the mode of action, we applied cyclic voltammetry and quartz crystal microbalance to correlate pore formation with lipid II binding kinetics of gallidermin in model membranes. The inability of gallidermin to form pores in DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) (C18/1) and DPoPC (1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine) (C16/1) membranes was related to the membrane thickness. For a better simulation of bacterial membrane characteristics, two different phospholipids with branched fatty acids were incorporated into the DPoPC matrix. Phospholipids with methyl branches in the middle of the fatty acid chains favored a lipid II–independent DPoPC permeabilization by gallidermin, while long-branched phospholipids in which the branch is placed near the hydrophilic region induced an identical lipid II–dependent pore formation of gallidermin and nisin. Obviously, the branched lipids altered lipid packing and reduced the membrane thickness. Therefore, the duality of gallidermin activity (pore formation and inhibition of the cell wall synthesis) seems to be balanced by the bacterial membrane composition.     

On the mechanism of pore formation by melittin

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

Energy-independent translocation of cell-penetrating peptides occurs without formation of pores. A biophysical study with pep-1

Pep-1 is a cell-penetrating peptide (CPP) with the ability to translocate across biological membranes and introduce active proteins inside cells. The uptake mechanism used by this CPP is, as yet, unknown in detail. Previous results show that such a mechanism is endocytosis-independent and suggests that physical-chemical interactions between the peptide and lipid bilayers govern the translocation mechanism. Formation of a transmembrane pore has been proposed but this issue has always remained controversial. In this work the secondary structure of pep-1 in the absence/presence of lipidic bilayers was determined by CD and ATR-FTIR spectroscopies and the occurrence of pore formation was evaluated through electrophysiological measurements with planar lipid membranes and by confocal microscopy using giant unilamellar vesicles. Despite pep-1 hydrophobic domain tendency for amphipathic α-helix conformation in the presence of lipidic bilayers, there was no evidence for membrane pores in the presence of pep-1. Furthermore, alterations in membrane permeability only occurred for high peptide/lipid ratios, which induced the complete membrane disintegration. Such observations indicate that electrostatic interactions are of first importance in the pep-1-membrane interactions and show that pores are not formed. A peptide-lipid structure is probably formed during peptide partition, which favours peptide translocation.     

Energy-independent translocation of cell-penetrating peptides occurs without formation of pores. A biophysical study with pep-1

Pep-1 is a cell-penetrating peptide (CPP) with the ability to translocate across biological membranes and introduce active proteins inside cells. The uptake mechanism used by this CPP is, as yet, unknown in detail. Previous results show that such a mechanism is endocytosis-independent and suggests that physical-chemical interactions between the peptide and lipid bilayers govern the translocation mechanism. Formation of a transmembrane pore has been proposed but this issue has always remained controversial. In this work the secondary structure of pep-1 in the absence/presence of lipidic bilayers was determined by CD and ATR-FTIR spectroscopies and the occurrence of pore formation was evaluated through electrophysiological measurements with planar lipid membranes and by confocal microscopy using giant unilamellar vesicles. Despite pep-1 hydrophobic domain tendency for amphipathic alpha-helix conformation in the presence of lipidic bilayers, there was no evidence for membrane pores in the presence of pep-1. Furthermore, alterations in membrane permeability only occurred for high peptide/lipid ratios, which induced the complete membrane disintegration. Such observations indicate that electrostatic interactions are of first importance in the pep-1-membrane interactions and show that pores are not formed. A peptide-lipid structure is probably formed during peptide partition, which favours peptide translocation.     

Transmembrane pore formation by the carboxyl terminus of Bax protein

Bax is a cytosolic protein that responds to various apoptotic signals by binding to the outer mitochondrial membrane, resulting in membrane permeabilization, release of cytochrome c, and caspase-mediated cell death. Currently discussed mechanisms of membrane perforation include formation of hetero-oligomeric complexes of Bax with other pro-apoptotic proteins such as Bak, or membrane insertion of multiple hydrophobic helices of Bax, or formation of lipidic pores physically aided by mitochondrial membrane-inserted proteins. There is compelling evidence provided by our and other groups indicating that the C-terminal “helix 9” of Bax mediates membrane binding and pore formation, yet the mechanism of pore forming capability of Bax C-terminus remains unclear. Here we show that a 20-amino acid peptide corresponding to Bax C-terminus (VTIFVAGVLTASLTIWKKMG) and two mutants where the two lysines are replaced with glutamate or leucine have potent membrane pore forming activities in zwitterionic and anionic phospholipid membranes. Analysis of the kinetics of calcein release from lipid vesicles allows determination of rate constants of pore formation, peptide–peptide affinities within the membrane, the oligomeric state of transmembrane pores, and the importance of the lysine residues. These data provide insight into the molecular details of membrane pore formation by a Bax-derived peptide and open new opportunities for design of peptide-based cytotoxic agents.     

Biophysical studies of a transmembrane peptide derived from the T cell antigen receptor

Summary Core peptide (CP) is a unique peptide derived from the transmembrane sequence of T cell antigen receptor (TCR)-alpha chain and is capable of inhibiting the immune response both invitro and in animal models of T cell mediated inflammation. The structure of CP, with sequence GLRILLLKV, is similar to the amphipathic region of many peptides. Unlike antimicrobial peptides, however, which damage cell membranes, electron microscopy and propidium iodide exclusion assays on cell membranes suggest that CP does not create pores and may act by interfering with signal transduction at the membrane level. To investigate this effect further we report the results of³¹P and²H solid-state NMR spectroscopy of CP on model membranes. As predicted, even at high concentrations of CP, the structure of model membranes was not significantly perturbed. Only at the very high peptide-to-lipid molar ratio of 1∶10 significant effects on the model membranes were observed. We conclude that CP does not destroy the integrity of the lipid bilayer.     

Alternative Mechanisms for the Interaction of the Cell-Penetrating Peptides Penetratin and the TAT Peptide with Lipid Bilayers

Cell-penetrating peptides (CPPs) have recently attracted much interest due to their apparent ability to penetrate cell membranes in an energy-independent manner. Here molecular-dynamics simulation techniques were used to study the interaction of two CPPs: penetratin and the TAT peptide with 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) phospolipid bilayers shed light on alternative mechanisms by which these peptides might cross biological membranes. In contrast to previous simulation studies of charged peptides interacting with lipid bilayers, no spontaneous formation of transmembrane pores was observed. Instead, the simulations suggest that the peptides may enter the cell by micropinocytosis, whereby the peptides induce curvature in the membrane, ultimately leading to the formation of small vesicles within the cell that encapsulate the peptides. Specifically, multiple peptides were observed to induce large deformations in the lipid bilayer that persisted throughout the timescale of the simulations (hundreds of nanoseconds). Pore formation could be induced in simulations in which an external potential was used to pull a single penetratin or TAT peptide into the membrane. With the use of umbrella-sampling techniques, the free energy of inserting a single penetratin peptide into a DPPC bilayer was estimated to be ∼75 kJmol⁻¹, which suggests that the spontaneous penetration of single peptides would require a timescale of at least seconds to minutes. This work also illustrates the extent to which the results of such simulations can depend on the initial conditions, the extent of equilibration, the size of the system, and the conditions under which the simulations are performed. The implications of this with respect to the current systems and to simulations of membrane-peptide interactions in general are discussed.     

Thermodynamic profiling of peptide membrane interactions by isothermal titration calorimetry: a search for pores and micelles

Antimicrobial peptides are known to interact strongly with negatively charged lipid membranes, initially by peripheral insertion of the peptide into the bilayer, which for some antimicrobial peptides will be followed by pore formation, and successive solubilization of the membranes resulting in mixed peptide-lipid micelles. We have investigated the mode of action of the antimicrobial peptide mastoparan-X using isothermal titration calorimetry (ITC) and cryo-transmission electron microscopy (cryo-TEM). The results show that mastoparan-X induces a range of structural transitions of POPC/POPG (3:1) lipid membranes at different peptide/lipid ratios. It has been established that ITC can be used as a fast method for localizing membrane transitions and when combined with DLS and cryo-TEM can elucidate structural changes, including the threshold for pore formation and micellation. Cryo-TEM was employed to confirm the structural changes associated with the thermodynamic transitions found by ITC. The pore-formation process has furthermore been investigated in detail and the thermodynamic parameters of pore formation have been characterized using a system-specific temperature where the enthalpy of peptide partitioning becomes zero (T_zero). This allows for an exclusive study of the pore-formation process. The use of ITC to find T_zero allows for characterization of the thermodynamic parameters of secondary processes on lipid membranes.